Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains

ABSTRACT

Novel polypeptides having the scaffold structure of a C-type lectin-like domain (CTLD) and a randomized loop region for specifically binding a variety of target compounds and also provides nucleic acids encoding the polypeptides. Combinatorial CTLD libraries, methods for constructing the libraries, and methods for screening the libraries to identify and isolate the novel CTLD polypeptides. Libraries of nucleic acids encoding polypeptides having a scaffold CTLD with a randomized loop region, as well as nucleic acid sequences, vectors, and methods for preparing and expressing the libraries. Exemplary nucleic acids useful in the combinatorial libraries are derived from tetranectin and other proteins having a CTLD.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/633040, filed Dec. 4, 2006, now U.S. Pat. No. 8,017,559, which is adivisional of U.S. patent application Ser. No. 10/450,472, filed Jun.13, 2003, which is a national phase application of InternationalApplication PCT/DK01/00825, filed Dec. 13, 2001, which claims priorityto Denmark application PA 2000 01872, filed Dec. 13, 2000 and U.S.Application No. 60/272,098, filed Feb. 28, 2001. The entire contents ofthe above-referenced applications are hereby incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

This invention describes a system which relates to the generation ofrandomized libraries of ligand-binding protein units derived fromproteins containing the so-called C-type lectin like domain (CTLD) ofwhich the carbohydrate recognition domain (CRD) of C-type lectinsrepresents one example of a family of this protein domain.

BACKGROUND OF THE INVENTION

The C-type lectin-like domain (CTLD) is a protein domain family whichhas been identified in a number of proteins isolated from many animalspecies (reviewed in Drickamer and Taylor (1993) and Drickamer (1999)).Initially, the CTLD domain was identified as a domain common to theso-called C-type lectins (calcium-dependent carbohydrate bindingproteins) and named “Carbohydrate Recognition Domain” (“CRD”). Morerecently, it has become evident that this domain is shared among manyeukaryotic proteins, of which several do not bind sugar moieties, andhence, the canonical domain has been named as CTLD.

CTLDs have been reported to bind a wide diversity of compounds,including carbohydrates, lipids, proteins, and even ice [Aspberg et al.(1997), Bettler et al. (1992), Ewart et al. (1998), Graversen et al.(1998), Mizumo et al. (1997), Sano et al. (1998), and Tormo et al.(1999)]. Only one copy of the CTLD is present in some proteins, whereasother proteins contain from two to multiple copies of the domain. In thephysiologically functional unit multiplicity in the number of CTLDs isoften achieved by assembling single copy protein protomers into largerstructures.

The CTLD consists of approximately 120 amino acid residues and,characteristically, contains two or three intra-chain disulfide bridges.Although the similarity at the amino acid sequence level between CTLDsfrom different proteins is relatively low, the 3D-structures of a numberof CTLDs have been found to be highly conserved, with the structuralvariability essentially confined to a so-called loop-region, oftendefined by up to five loops. Several CTLDs contain either one or twobinding sites for calcium and most of the side chains which interactwith calcium are located in the loop-region.

On the basis of CTLDs for which 3D structural information is available,it has been inferred that the canonical CTLD is structurallycharacterised by seven main secondary-structure elements (i.e. fiveβ-strands and two α-helices) sequentially appearing in the order β1; α1;α2; β2; β3; β4; and β5 (FIG. 1, and references given therein). In allCTLDs, for which 3D structures have been determined, the β-strands arearranged in two anti-parallel β-sheets, one composed of β1 and β5, theother composed of β2, β3 and β4. An additional β-strand, β0, oftenprecedes β1 in the sequence and, where present, forms an additionalstrand integrating with the β1, β5-sheet. Further, two disulfidebridges, one connecting α1 and β5 (C_(I)-C_(IV), FIG. 1) and oneconnecting β3 and the polypeptide segment connecting β4 and β5(C_(II)-C_(III), FIG. 1) are invariantly found in all CTLDscharacterised so far. In the CTLD 3D-structure, these conservedsecondary structure elements form a compact scaffold for a number ofloops, which in the present context collectively are referred to as the“loop-region”, protruding out from the core. These loops are in theprimary structure of the CTLDs organized in two segments, loop segmentA, LSA, and loop segment B, LSB. LSA represents the long polypeptidesegment connecting β2 and β3 which often lacks regular secondarystructure and contains up to four loops. LSB represents the polypeptidesegment connecting the β-strands β3 and β4. Residues in LSA, togetherwith single residues in β4, have been shown to specify the Ca²⁺- andligand-binding sites of several CTLDs, including that of tetranectin.E.g. muta-genesis studies, involving substitution of single or a fewresidues, have shown, that changes in binding specificity,Ca²⁺-sensitivity and/or affinity can be accommodated by CTLD domains[Weis and Drickamer (1996), Chiba et al. (1999), Graversen et al.(2000)].

As noted above, overall sequence similarities between CTLDs are oftenlimited, as assessed e.g. by aligning a prospective CTLD sequence withthe group of structure-characterized CTLDs presented in FIG. 1, usingsequence alignment procedures and analysis tools in common use in thefield of protein science. In such an alignment, typically 22-30% of theresidues of the prospective CTLD will be identical with thecorresponding residue in at least one of the structure-characterizedCTLDs. The sequence alignment shown in FIG. 1 was strictly elucidatedfrom actual 3D structure data, so the fact that the polypeptide segmentsof corresponding structural elements of the framework also exhibitstrong sequence similarities provide a set of direct sequence-structuresignatures, which can readily be inferred from the sequence alignment.

The implication is that also CTLDs, for which precise 3D structuralinformation is not yet available, can nonetheless be used as frameworksin the construction of new classes of CTLD libraries. The specificadditional steps involved in preparing starting materials for theconstruction of such a new class of CTLD library on the basis of a CTLD,for which no precise 3D structure is available, would be the following:(1) Alignment of the sequence of the new CTLD with the sequence shown inFIG. 1; and (2) Assignment of approximate locations of frameworkstructural elements as guided by the sequence alignment, observing anyrequirement for minor adjustment of the alignment to ensure precisealignment of the four canonical cysteine residues involved in theformation of the two conserved disulfide bridges (C_(I)-C_(IV) andC_(II)-C_(III), in FIG. 1). The main objective of these steps would beto identify the sequence location of the loop-region of the new CTLD, asflanked in the sequence by segments corresponding to the β2-, β3-, andβ4-strands. To provide further guidance in this the results of ananalysis of the sequences of 29 bona fide CTLDs are given in Table 1below in the form of typical tetrapeptide sequences, and their consensussequences, found as parts of CTLD β2- and β3-strands, and the preciselocation of the β4-strand by position and sequence characteristics aselucidated.

TABLE I β2 and β3 consensus elements analysis                                                                                                                                                       SEQ                                                                                                                                                        ID CTLD β2                    ---                           LSA NO IX-AW I G L R W - - - Q G KVKQCNS E W S D G S S V S - - Y E N W I E - - - - - - - - 92 MGLW I G L T D Q - - N G P - - W R W V D G T D F E K G F K N W A P - - - - - - - - 93 LITW I G L H D P K K N R R - - W H W S S G S L V S - - Y K S W G I - - - - - - - - 94 CHLW I G L T D E N Q E G E - - W Q W V D G T D T R S S F T F W K E - - - - - - - - 95 IGE-W I G L R N L D L K G E F I W V - - D G S H V D - - Y S N W A P - - - - - - - - 96 FCR TCL-1W I G L T D K D S E G T - - W K W V D G T P L T - - T A F W S T - - - - - - - - 97 KUCRW I G L T D Q G T E G N - - W R W V D G T P F DYVQS R R F W R K - - - - - - - - 98 CD94W I G L S Y S E E H T A - - W L W E N G S A L S Q - Y L S F E T - - - - - - - - 99 CPCPW I G L N D R T I E G D F R W S - - D G H P M Q - - F E N W R P - - - - - - - -100 PAPW I G L H DPTQGTEPN G E G - W E W S S S D V M N - - Y F A W E R - - - - - - - -101 NEUW I G L N D R I V E Q D - - F Q W T D N T G L Q - - Y E N W R E - - - - - - - -102 ESLW I G I R K V N N V - - - - W V W - V G T Q K P L T EEAKN W A P - - - - - - - -103 NKg2AW I G V F R N S S H H P - - W V T M N G L A F K H E I K D S D N A - - - - - - -104 GP120W M G L S D L N Q E G T - - W Q W V D G S PLL P S - FKQ Y W N R - - - - - - - -105 MRW I G L F R N V - E G T - - W L W I N N S P V S - - F V N W N T - - - - - - - -106 TNW L G L N D M A A E G T - - - - W V D M T G A R I A Y K N W E T E I T - - - - -107 SCGFW L G V H D R R A E G L - - Y L F E N G Q R V S - - F F A W HRSPRPELGAQPSASPHPLS108 PLCW L G A S D L N I E G R - - W L W - E G Q R R M N - Y T N W S P - - - - - - - -109 H1-W M G L H D - - Q N G P - - W K W V D G T D Y E T G F K N W R P - - - - - - - -110 ASR IX-BW M G L S N V W N Q C N - - W Q W S N A A M L R - - Y K A W A E - - - - - - - -111 LY49AW V G L S Y D N K K K D - - W A W I D N R P S K L A L N T R K Y - - - - - - - -112 TU14W V G A D N - L Q D G A Y N F N W N D G V S L P T D S D L W S P - - - - - - - -113 rSP-AY L G M I E D Q T P G D - - F H Y L D G A S V N - - Y T N W Y P - - - - - - - -114 BCONY L S M N D I S T E G R - - F T Y P T G E I L V - - Y S N W A D - - - - - - - -115 BCL43Y L S M N D I S K E G K - - F T Y P T G G S L D - - Y S N W A P - - - - - - - -116 MBP-AF L G I T D E V T E G Q - - F M Y V T G G R L T - - Y S N W K K - - - - - - - -117 SP-DF L S M T D S K T E G K - - F T Y P T G E S L V - - Y S N W A P - - - - - - - -118 CL-L1F I G V N D L E R E G Q - - Y M F T D N T P L Q N - Y S N W N E - - - - - - - -119 DCIRF V G L S D P - - E G Q R H W Q W V D Q T P - - - - Y NESSTFWHP - - - - - - - -120   SEQ ID CTLD  ---                           β3        LSB         β4 NO IX-AA E S K T - - - - - - - - - - - C L G L E KET D F R K W V N I Y C  92MGL L Q P D N W F G H G L G G G E D C A H I T T G - - G F W N D D V C 93 LITG A P S S V N P - - - - - G Y - C V S L TSS T G F Q K W K D V P C  94CHL G E P N N R G F - - - - - N E D C A H V W T S - - G Q W N D V Y C 95 IGE-G E P T S R S Q - - - - - G E D C V M M R G S - - G R W N D A F C  96FCR TCL-1D E P N D G A V N - - - - G E D C V S L Y YHTQPEF K N W N D L A C  97KUCR G Q P D W R H G N G E - - R E D C V H L Q - - - - R M W N D M A C 98 CD94- - - - F N T K N - - - - - - - C I A Y N P N - - G N A L D E S C  99CPCP N Q P D N F F A A - - - - G E D C V V M I W H E K G E W N D V P C100 PAPN - P S T I S S P G H - - - - - C A S L S RST A F L R W K D Y N C 101NEU N Q P D N F F A G - - - - G E D C V V L V S H E I G K W N D V P C102 ESLG E P N N R Q K - - - - - D E D C V E I YIKREKD V G M W N D E R C 103NKg2A - - - - - - - - - - - - - E L N C A V L Q V - - - N R L K S A Q C104 GP120G E P N N V G - - - - - - E E D C A E F S G N - - G - W N D D K C 105 MRG D P S G E - - - - - - - R N D C V A L H A S S - G F W S N I H C 106 TNA Q P D G G K - - - - - - T E N C A V L S G A A N G K W F D K R C 107SCGF PDQ P N G G T - - - - - - L E N C V A Q A S D D - G S W W D H D C108 PLCG Q P D N A G G - - - - - I E H C L E L RRD L G N Y L W N D Y Q C 109H1- E Q P D D W Y G H G L G G G E D C A H F T D D - - G R W N D D V C110 ASR IX-BE S Y - - - - - - - - - - - - - C V Y F K S T N - N K W R S R A C 111LY49A N I R D G G - - - - - - - - - - C M L L S K T - - - R L D N G N C112 TU14N E P S N P Q S W Q L - - - - - C V Q I W S K Y - N L L D D V G C 113rSP-A G E P R G Q G - - - - - - K E K C V E M Y T D - - G T W N D R G C114 BCONG E P N N S D E G Q - - - P E N C V E I F P D - - G K W N D V P C 115BCL43 G E P N N R A K D E G - - P E N C L E I Y S D - - G N W N D I E C116 MBP-AD E P N D H G S - - - - - G E D C V T I V D N - - G L W N D I S C 117SP-D G E P N D D G G - - - - - S E D C V E I F T N - - G K W N D R A C118 CL-L1G E P S D P Y G - - - - - H E D C V E M L S S - - G R W N D T E C 119DCIR R E P S D P N - - - - - - - E R C V V L NFRKSPKRW G - W N D V N C120 Notes: LSA, Loop Segment A; LSB, Loop Segemnt B.

Sequences taken from: Berglund and Petersen (1992) [TN, tetranectin];Bertrand et al. (1996) [LIT, lithostatin]; Mann et al. (2000) [MGL,mouse macrophage galactose lectin, KUCR, Kupffer cell receptor, NEU,chicken neurocan, PLC, perlucin, H1-ASR, asialoglycoprotein receptor];Mio et al. (1998) [CPCP, cartilage proteoglycan core protein, IGE-FCR,IgE Fc receptor, PAP, pancreatitis-associated protein, MMR, mousemacrophage receptor, NKG2, Natural Killer group, SCGF, stem cell growthfactor]; Mizuno et al. (1997) [IX-A and B, factor IX/X binding protein,MBP, mannose binding protein]; Ohtani et al. (1999) [BCON, bovineconglutinin, BCL43, bovine CL43, CL-L1, collectin liver 1, SP-A,surfactant protein A, SP-D, surfactant protein D]; Poget et al. (1999)[ESL, e-selectin, TU14, tunicate c-type lectin]; Tormo et al. (1999)[CD94,CD94 NK receptor domain, LY49A, LY49A NK receptor domain]; Zhanget al. (2000) [CHL, chicken hepatic lectin, TCL-1, trout c-type lectin,GP120, HIV gp 120-binding c-type lectin, DCIR, dendritic cell immunoreceptor]

Of the 29 β2-strands,

-   -   14 were found to conform to the consensus sequence WIGX (SEQ ID        NO: 305) (of which 12 were WIGL (SEQ ID NO: 306) sequences, 1        was a WIGI (SEQ ID NO: 307) sequence and 1 was a WIGV (SEQ ID        NO: 308) sequence);    -   3 were found to conform to the consensus sequence WLGX (SEQ ID        NO. 309) (of which 1 was a WLGL (SEQ ID NO: 310) sequence, 1 was        a WLGV (SEQ ID NO: 311) sequence and 1 was a WLGA (SEQ ID        NO: 312) sequence);    -   3 were found to be WMGL (SEQ ID NO: 313) sequences;    -   3 were found to conform to the consensus sequence YLXM (SEQ ID        NO: 314)(of which 2 were YLSM (SEQ ID NO:315) sequences and 1        was an YLGM (SEQ ID NO: 316) sequence);    -   2 were found to conform to the consensus sequence WVGX (SEQ ID        NO: 317] (of which 1 was a WVGL (SEQ ID NO: 318] sequence and 1        was a WVGA (SEQ ID NO: 319] sequence); and    -   the sequences of the remaining 4 β2-strands in the collection        were FLGI (SEQ ID NO: 320), FVGL (SEQ ID NO: 321), FIGV (SEQ ID        NO: 322) and FLSM {SEQ ID NO: 323) sequences, respectively.

Therefore, it is concluded that the four-residue β2 consensus sequence(“β2cseq”) may be specified as follows:

-   -   Residue 1: An aromatic residue, most preferably Trp, less        preferably Phe and least preferably Tyr.    -   Residue 2: An aliphatic or non-polar residue, most preferably        Ile, less preferably Leu or Met and least preferably Val.    -   Residue 3: An aliphatic or hydrophilic residue, most preferably        Gly and least preferably Ser.    -   Residue 4: An aliphatic or non-polar residue, most preferably        Leu and less preferably Met, Val or Ile.

Accordingly the P2 consensus sequence may be summarized as follows:

-   -   β2cseq: (W,Y,F)-(I,L,V,M)-(G,S)-(L,M,V,I),    -   where the underlined residue denotes the most commonly found        residue at that sequence position.

All 29 β3-strands analyzed are initiated with the Cys_(II) residuecanonical for all known CTLD sequences, and of the 29 β3-strands,

-   -   5 were found to conform to the consensus sequence CVXI (SEQ ID        NO: 324) (of which 3 were CVEI (SEQ ID NO: 325) sequences, 1 was        a CVTI (SEQ ID NO: 326) sequence and 1 was a CVQI (SEQ ID        NO: 327) sequence);    -   4 were found to conform to the consensus sequence CVXM (SEQ ID        NO: 328) (of which 2 were CVEM (SEQ ID NO: 329) sequences, 1 was        a CVVM (SEQ ID NO: 330) sequence and 1 was a CVMM (SEQ ID        NO: 331) sequence);    -   6 were found to conform to the consensus sequence CVXL (SEQ ID        NO: 332) (of which 2 were CVVL (SEQ ID NO: 333) sequences, 2        were a CVSL (SEQ ID NO: 334 sequence, 1 was a CVHL (SEQ ID        NO: 335) sequence and 1 was CVAL (SEQ ID NO: 336) sequence);    -   3 were found to conform to the consensus sequence CAXL (SEQ ID        NO: 337) (of which 2 were CAVL (SEQ ID NO: 338) sequences and 1        was a CASL (SEQ ID NO: 339) sequence);    -   2 were found to conform to the consensus sequence CAXF (SEQ ID        NO: 340) (of which 1 was 1 CAHF (SEQ ID NO: 341) sequence and 1        was a CAEF (SEQ ID NO: 342) sequence);    -   2 were found to conform to the consensus sequence CLXL (SEQ ID        NO: 343) (of which 1 was a CLEL (SEQ ID NO: 344) sequence and 1        was a CLGL (SEQ ID NO: 345) sequence); and    -   the sequences of the remaining 7 β3-strands in the collection        were CVYF (SEQ ID NO: 346), CVAQ (SEQ ID NO: 347), CAHV (SEQ ID        NO: 348), CAHI (SEQ ID NO:349), CLEI (SEQ ID NO: 350), CIAY (SEQ        ID NO: 351), and CMLL (SEQ ID NO: 352) sequences, respectively.

Therefore, it is concluded that the four-residue β3 consensus sequence(“(33cseq”) may be specified as follows:

-   -   Residue 1: Cys, being the canonical Cys_(II) residue of CTLDs    -   Residue 2: An aliphatic or non-polar residue, most preferably        Val, less preferably Ala or Leu and least preferably Ile or Met    -   Residue 3: Most commonly an aliphatic or charged residue, which        most preferably is Glu    -   Residue 4: Most commonly an aliphatic, non-polar, or aromatic        residue, most preferably Leu or Ile, less preferably Met or Phe        and least preferably Tyr or Val.

Accordingly the β3 consensus sequence may be summarized as follows:

-   -   β3cseq: (C)-(V,A,L,I,M)-(E,X)-(L,I,M,F,Y,V),    -   where the underlined residue denotes the most commonly found        residue at that sequence position.

It is observed from the known 3D-structures of CTLDs (FIG. 1), that theβ4-strands most often are comprised by five residues located in theprimary structure at positions −6 to −2 relative to the canonicalCys_(III) residue of all known CTLDs, and less often are comprised byfour residues located at positions −5 to −2 relative to the canonicalCys_(III) residue of all known CTLDs. The residue located at position−3, relative to Cys_(III), is involved in co-ordination of the site 2calcium ion in CTLDs housing this site, and this notion is reflected inthe observation, that of the 29 CTLD sequences analyzed in Table 1, 27have an Asp-residue or an Asn-residue at this position, whereas 2 CTLDshave a Ser at this position. From the known CTLD 3D-structures it isalso noted, that the residue located at position −5, relative to theCys_(III) residue, is involved in the formation of the hydrophobic coreof the CTLD scaffold. This notion is reflected in the observation, thatof the 29 CTLD sequences analyzed 25 have a Trp-residue, 3 have aLeu-residue, and 1 an Ala-residue at this position. 18 of the 29 CTLDsequences analyzed have an Asn-residue at position −4. Further, 19 ofthe 29 β4-strand segments are preceded by a Gly residue.

Of the 29 central three residue motifs located at positions −5, −4 and−3 relative to the canonical Cys_(III) residue in the β4-strand:

-   -   22 were of the sequence WXD (18 were WND, 2 were WKD, 1 was WFD        and 1 was WWD),    -   2 were of the sequence WXN (1 was WVN and 1 was WSN),    -   and the remaining 5 motifs (WRS, LDD, LDN, LKS and ALD) were        each represented once in the analysis.

It has now been found that each member of the family of CTLD domainsrepresents an attractive opportunity for the construction of new proteinlibraries from which members with affinity for new ligand targets can beidentified and isolated using screening or selection methods. Suchlibraries may be constructed by combining a CTLD framework structure inwhich the CTLD's loop-region is partially or completely replaced withone or more randomized polypeptide segments.

One such system, where the protein used as scaffold is tetranectin orthe CTLD domain of tetranectin, is envisaged as a system of particularinterest, not least because the stability of the trimeric complex oftetranectin protomers is very high (International Patent ApplicationPublication No. WO 98/56906 A2).

Tetranectin is a trimeric glycoprotein [Holtet et al. (1997), Nielsen etal. (1997)], which has been isolated from human plasma and found to bepresent in the extracellular matrix in certain tissues. Tetranectin isknown to bind calcium, complex polysaccharides, plasminogen,fibrinogen/fibrin, and apolipoprotein (a). The interaction withplasminogen and apolipoprotein (a) is mediated by the so-called kringle4 protein domain therein. This interaction is known to be sensitive tocalcium and to derivatives of the amino acid lysine [Graversen et al.(1998)].

A human tetranectin gene has been characterised, and both human andmurine tetranectin cDNA clones have been isolated. Both the human andthe murine mature protein comprise 181 amino acid residues (FIG. 2). The3D-structures of full length recombinant human tetranectin and of theisolated tetranectin CTLD have been determined independently in twoseparate studies [Nielsen et al. (1997) and Kastrup et al. (1998)].Tetranectin is a two- or possibly three-domain protein, i.e. the mainpart of the polypeptide chain comprises the CTLD (amino acid residuesGly53 to Val181), whereas the region Leu26 to Lys52 encodes analpha-helix governing trimerisation of the protein via the formation ofa homotrimeric parallel coiled coil. The polypeptide segment Glu1 toGlu25 contains the binding site for complex polysaccharides (Lys6 toLys15) [Lorentsen et al. (2000)] and appears to contribute tostabilization of the trimeric structure [Holtet et al. (1997)]. The twoamino acid residues Lys148 and Glu150, localized in loop 4, and Asp165(localized in β4) have been shown to be of critical importance forplasminogen kringle 4 binding, whereas the residues Ile140 (in loop 3)and Lys166 and Arg167 (in β4) have been shown to be of some importance[Graversen et al. (1998)]. Substitution of Thr149 (in loop 4) with anaromatic residue has been shown to significantly increase affinity oftetranectin to kringle 4 and to increase affinity for plasminogenkringle 2 to a level comparable to the affinity of wild type tetranectinfor kringle 4 [Graversen et al. (2000)].

OBJECT OF THE INVENTION

The object of the invention is to provide a new practicable method forthe generation of useful protein products endowed with binding sitesable to bind substance of interest with high affinity and specificity.

The invention describes one way in which such new and useful proteinproducts may advantageously be obtained by applying standardcombinatorial protein chemistry methods, commonly used in therecombinant antibody field, to generate randomized combinatoriallibraries of protein modules, in which each member contains anessentially common core structure similar to that of a CTLD.

The variation of binding site configuration among naturally occurringCTLDs shows that their common core structure can accommodate manyessentially different configurations of the ligand binding site. CTLDsare therefore particularly well suited to serve as a basis forconstructing such new and useful protein products with desired bindingproperties.

In terms of practical application, the new artificial CTLD proteinproducts can be employed in applications in which antibody products arepresently used as key reagents in technical biochemical assay systems ormedical in vitro or in vivo diagnostic assay systems or as activecomponents in therapeutic compositions.

In terms of use as components of in vitro assay systems, the artificialCTLD protein products are preferable to antibody derivatives as eachbinding site in the new protein product is harboured in a singlestructurally autonomous protein domain. CTLD domains are resistant toproteolysis, and neither stability nor access to the ligand-binding siteis compromised by the attachment of other protein domains to the N- orC-terminus of the CTLD. Accordingly, the CTLD binding module may readilybe utilized as a building block for the construction of modularmolecular assemblies, e.g. harbouring multiple CLTDs of identical ornonidentical specificity in addition to appropriate reporter moduleslike peroxidases, phosphatases or any other signal-mediating moiety.

In terms of in vivo use as essential component of compositions to beused for in vivo diagnostic or therapeutic purposes, artificial CTLDprotein products constructed on the basis of human CTLDs are virtuallyidentical to the corresponding natural CTLD protein already present inthe body, and are therefore expected to elicit minimal immunologicalresponse in the patient. Single CTLDs are about half the mass of thesmallest functional antibody derivative, the single-chain Fv derivative,and this small size may in some applications be advantageous as it mayprovide better tissue penetration and distribution, as well as a shorterhalf-life in circulation. Multivalent formats of CTLD proteins, e.g.corresponding to the complete tetranectin trimer or the furthermultimerized collectins, like e.g. mannose binding protein, provideincreased binding capacity and avidity and longer circulation half-life.

One particular advantage of the preferred embodiment of the invention,arises from the fact that mammalian tetranectins, as exemplified bymurine and human tetranectin, are of essentially identical structure.This conservation among species is of great practical importance as itallows straightforward swapping of polypeptide segments definingligand-binding specificity between e.g. murine and human tetranectinderivatives. The option of facile swapping of species genetic backgroundbetween tetranectin derivatives is in marked contrast to the well-knowncomplications of effecting the “humanisation” of murine antibodyderivatives.

Further advantages of the invention are:

The availability of a general and simple procedure for reliableconversion of an initially selected protein derivative into a finalprotein product, which without further reformatting may be produced inbacteria (e.g. Escherichia coli) both in small and in large scale(International Patent Application Publication No. WO 94/18227 A2).

The option of including several identical or non-identical binding sitesin the same functional protein unit by simple and general means, therebyenabling the exploitation even of weak affinities by means of avidity inthe interaction, or the construction of bi- or heterofunctionalmolecular assemblies (International Patent Application Publication No.WO 98/56906 A2).

The possibility of modulating binding by addition or removal of divalentmetal ions (e.g. calcium ions) in combinational libraries with one ormore preserved metal binding site(s) in the CTLDs.

SUMMARY OF THE INVENTION

The present invention provides a great number of novel and usefulproteins each being a protein having the scaffold structure of C-typelectin-like domains (CTLD), said protein comprising a variant of a modelCTLD wherein the α-helices and β-strands and connecting segments areconserved to such a degree that the scaffold structure of the CTLD issubstantially maintained, while the loop region is altered by amino acidsubstitution, deletion, insertion or any combination thereof, with theproviso that said protein is not any of the known CTLD loop derivativesof C-type lectin-like proteins or C-type lectins listed in the followingTable 2.

TABLE 2 LSA derivatives (β2 and β3 consensus elements are underlined)SEQ ID CTLD Mut. LSA sequence (one letter code) Reference NO hTN TND116AW L G L N A M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 121 T A Q P D G G K T E N C A V L (1998) TNE120AW L G L N D M A A A G T W V D M T G A R I A Y K N W E T E IGraversen et al. 122 T A Q P D G G K T E N C A V L (1998) TNK134AW L G L N D M A A E G T W V D M T G A R I A Y A N W E T E IGraversen et al. 123 T A Q P D G G K T E N C A V L (1998) TNI140AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E AGraversen et al. 124 T A Q P D G G K T E N C A V L (1998) TNQ143AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 125 T A A P D G G K T E N C A V L (1998) TND145AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 126 T A Q P A G G K T E N C A V L (1998) TNK148AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 127 T A Q P D G G A T E N C A V L (1998) TNK148MW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 128 T A Q P D G G M T E N C A V L (2000) TNK148RW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 129 T A Q P D G G R T E N C A V L (2000) TNT149FW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 130 T A Q P D G G K F E N C A V L (2000) TNT149MW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 131 T A Q P D G G K M E N C A V L (2000) TNT149RW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 132 T A Q P D G G K R E N C A V L (2000) TNT149YW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 133 T A Q P D G G K Y E N C A V L (2000) TNE150AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 134 T A Q P D G G K T A N C A V L (1998) TNE150DW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 135 T A Q P D G G K T D N C A V L (2000) TNE150QW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 136 T A Q P D G G K T Q N C A V L (2000) TNN151AW L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 137 T A Q P D G G K T E A C A V L (1998) TNK148R,W L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 138 T149Y T A Q P D G G R Y E N C A V L (2000) TNT149Y,W L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 139 E150Q T A Q P D G G K Y Q N C A V L (2000) TNT149Y,W L G L N D M A A E G T W V D M T G A R I A Y K N W E T E IGraversen et al. 140 D165N T A Q P D G G K Y E N C A V L (2000) rMBP QPDF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QDrickamer (1992) 141 P D D H G S G E D C V T I N187DF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D E Iobst et al.142 P D D H G S G E D C V T I (1994) H189AF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D E Iobst et al.143 P N D A G S G E D C V T I (1994) H189GF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D E Iobst et al.144 P N D G G S G E D C V T I (1994) QPDWF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 145 P D D W G S G E D C V T I (1994) QPDWGF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 146 P D D W Y G HGLGG G E D C V T I (1994) QPDWG/Y/AF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 147 P D D W A G HGLGG G E D C V T I (1994) QPDWG/Y/QF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 148 P D D W Q G HGLGG G E D C V T I (1994) QPDWG/G/AF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 149 P D D W Y A HGLGG G E D C V T I (1994) QPDWG/H/AF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 150 P D D W Y G AGLGG G E D C V T I (1994) QPDWG/H/QF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 151 P D D W Y G QGLGG G E D C V T I (1994) QPDWG/H/EF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 152 P D D W Y G EGLGG G E D C V T I (1994) QPDWG/H/YF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 153 P D D W Y G YGLGG G E D C V T I (1994) QPDWG/-/GF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 154 P D D W Y G HGL G G E D C V T I (1994) QPDFF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 155 P D D F G S G E D C V T I (1994) QPDFGF L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QIobst & Drickamer 156 P D D F Y G HGLGG G E D C V T I (1994) REGION 1F L G I R K V N N V F M Y V T G G R L T Y S N W K K D E P NBlanck et al. 157 D H G S G E D C V T I (1996) REGION 2F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D EBlanck et al. 158 P N N R Q K D E D C V T I (1996) RES. 189F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D ETorgersen et al. 159 P N D G G S G E D C V T I (1998) RES. 197F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D ETorgersen et al. 160 P N D H G S G E D C V E I (1998) LOOP 3EF L G I T D E V T E G Q F M Y V T G G R L T Y S N W A P G ETorgersen et al. 161 P N D H G S G E D C V T I (1998) LOOP 3PF L G I T D E V T E G Q F M Y V T G G R L T Y S N W A D N ETorgersen et al. 162 P N D H G S G E D C V T I (1998) REGION 4F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K K D QKolatkar et al. 163 P D D W Y G HGLGG G E D C V H I (1998) REGION 4'F L G I T D E V T E G Q F M Y V T G G R L T Y S N W R P G QKolatkar et al. 164 P D D W Y G HGLGG G E D C V H I (1998) QPDWG/QNGF L G I T D Q N G Q F M Y V T G G R L T Y S N W K K D Q P DWragg & Drickamer 165 D W Y G HGLGG G E D C V T I (1999) QPDWG/QNGPF L G I T D Q N G P F M Y V T G G R L T Y S N W K K D Q P DWragg & Drickamer 166 D W Y G HGLGG G E D C V T I (1999) MBP/CHL189F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K E G EBurrows et al. 167 P N N R G S G E D C V T I (1997) MBP/CHL192F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K E G EBurrows et al. 168 P N N R G F N E D C V T I (1997) MBP/CHL208F L G I T D E V T E G Q F M Y V T G G R L T Y S N W K E G EBurrows et al. 169 P N N R G F N E D C A H V (1997) rSP-A E195Q,Y L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G QMcCormack et al. 170 R197D P D G Q G K E K C V E M (1994) AM2Y L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G E Honma et al.171 P R G Q G K E K C V T I (1997) AM3Y L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G E Honma et al.172 P N D H G S G E D C V T I (1997) E195AY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G AMcCormack et al. 173 P R G Q G K E K C V E M (1997) R197GY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EMcCormack et al. 174 P G G Q G K E K C V E M (1997) E202AY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EMcCormack et al. 175 P R G Q G K A K C V E M (1997) N187SY L G M I E D Q T P G D F H Y L D G A S V S Y T N W Y P G EMcCormack et al. 176 P R G Q G K E K C V E M (1997) R197AY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EPattanajitvilai 177 P A G Q G K E K C V E M et al. (1998) R197KY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EPattanajitvilai 178 P K G Q G K E K C V E M et al. (1998) R197HY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EPattanajitvilai 179 P H G Q G K E K C V E M et al. (1998) R197DY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EPattanajitvilai  180 P D G Q G K E K C V E M et al. (1998) R197NY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G EPattanajitvilai 181 P N G Q G K E K C V E M et al. (1998) E195QY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G QTsunezawa et al. 182 P R G Q G K E K C V E M (1998) K201AY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G ETsunezawa et al. 183 P R G Q G A E K C V E M (1998) K203AY L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G ETsunezawa et al. 184 P R G Q G K E A C V E M (1998) E197A,Y L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G ATsunezawa et al. 185 K201A,K203A P R G Q G A E A C V E M (1998) ad3Y L G M I E D Q T P G D F H Y L D G A S V N Y T N W Y P G ESano et al. (1998) 186 P N N N G G A E N C V E I ad4Y L G M I E D Q T E G K F T Y P T G E A L V Y S N W A P G ESano et al. (1998) 187 P N N N G G A E N C V E I rat ama4Y L G M I E D Q T E G Q F M Y V T G G R L T Y S N W K K D EChiba et al (1999) 188 P R G Q G K E K C V E M hSP-A R199AY V G L T E G P S P G D F R Y S D G T P V N Y T N W Y R G ETsunezawa et al. 189 P A G A G K E Q C V E M (1998) K201AY V G L T E G P S P G D F R Y S D G T P V N Y T N W Y R G ETsunezawa et al. 190 P A G R G A E Q C V E M (1998) hum ama4Y V G L T E G P T E G Q F M Y V T G G R L T Y S N W K K D EChiba et al (1999) 191 P R G R G K E Q C V E M rSP-D E321Q,F L S M T D V G T E G K F T Y P T G E A L V Y S N W A P G QOgasawara & Voelker 192 N323D P D N N G G A E N C V E I (1995) h-eslK67A W I G I R K V N N V W V W V G T Q A P L T E E A K N W A P GErbe et al. 193 E P N N R Q K D E D C V E I K74AW I G I R K V N N V W V W V G T Q K P L T E E A A N W A P G Erbe et al.194 E P N N R Q K D E D C V E I R84A,K86AW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P G Erbe et al.195 E P N N A Q A D E D C V E I R84AW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 196 E P N N A Q K D E D C V E I R84KW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 197 E P N N K Q K D E D C V E I R84K,D89GW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 198 E P N N K Q K D E G C V E I A77KW I G I R K V N N V W V W V G T Q K P L T E E A K N W K P GKogan et al. (1995) 199 E P N N R Q K D E D C V E I A77K,P78KW I G I R K V N N V W V W V G T Q K P L T E E A K N W K K GKogan et al. (1995) 200 E P N N R Q K D E D C V E I A77K,P78K,W I G I R K V N N V W V W V G T Q K P L T E E A K N W K K GKogan et al. (1995) 201 R84A E P N N A Q K D E D C V E I D87EW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 202 E P N N R Q K E E D C V E I D87NW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 203 E P N N R Q K N E D C V E I D89NW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 204 E P N N R Q K D E N C V E I D89EW I G I R K V N N V W V W V G T Q K P L T E E A K N W A P GKogan et al. (1995) 205 E P N N R Q K D E E C V E I A77K,E80Q,W I G I R K V N N V W V W V G T Q K P L T E E A K N W K P GKogan et al. (1995) 206 N82D Q P D N R Q K D E D C V E I h-psl A77KW I G I R K N N K T W T W V G T K K A L T N E A E N W K D NRevelle et al.  207 E P N N K R N N E D C V E I (1996) A77K,E80D,W I G I R K N N K T W T W V G T K K A L T N E A E N W K D NRevelle et al.  208 N82D Q P D N K R N N E D C V E I (1996) MGR 2A/RWIGL T D Q N G P W R W V D G T D Y E K G F T H W R P K Q P Iobst & Drickamer 209 D N W Y G H G L G G G E D CAHF (1996) 2K/GWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P G Q P Iobst & Drickamer 210 D N W Y G H G L G G G E D CAHF (1996) 2A/R,2K/GWIGL T D Q N G P W R W V D G T D Y E K G F T H W R P G Q P Iobst & Drickamer 211 D N W Y G H G L G G G E D CAHF (1996) 4F/IWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 212 D N W Y G H G L G G G E D CAHI (1996) 4H/AWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 213 D N W Y G H G L G G G E D CAAF (1996) 4H/EWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 214 D N W Y G H G L G G G E D CAEF (1996) 4H/QWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 215 D N W Y G H G L G G G E D CAQF (1996) 4H/NWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 216 D N W Y G H G L G G G E D CANF (1996) 4H/YWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 217 D N W Y G H G L G G G E D CAYF (1996) 4H/DWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 218 D N W Y G H G L G G G E D CADF (1996) 4H/KWIGL T D Q N G P W R W V D G T D Y E K G F T H W A P K Q P Iobst & Drickamer 219 D N W Y G H G L G G G E D CAKF (1996) 2A/R,2K/G,WIGL T D Q N G P W R W V D G T D Y E K G F T H W R P G Q P Iobst & Drickamer 220 4H/A D N W Y G H G L G G G E D CAAF (1996) RHL4H/A WIGL T D Q N G P W K W V D G T D Y E T G F K N W R P G Q P Iobst & Drickamer 221 D D W Y G H G L G G G E D CAAF (1996) CHL R173AW I G L T D E N Q E G E W Q W V D G T D T R S S F T F W K EBurrows et al.  222 G E P N N A G F N E D C A H V (1997) G174AW I G L T D E N Q E G E W Q W V D G T D T R S S F T F W K EBurrows et al.  223 G E P N N R A F N E D C A H V (1997) F175AW I G L T D E N Q E G E W Q W V D G T D T R S S F T F W K EBurrows et al.  224 G E P N N R G A N E D C A H V (1997) N176AW I G L T D E N Q E G E W Q W V D G T D T R S S F T F W K EBurrows et al.  225 G E P N N R G F A E D C A H V (1997)LSB derivatives (β3 and β4 consensus elements are underlined) SEQ IDCTDL Mut. LSB sequence (one letter code) Reference NO hTN TNK163AC A V L S G A A N G A W F D K R C Graversen et al. 226 (1998) TNK166AC A V L S G A A N G K W F D A R C Graversen et al. 227 (1998) TNR167AC A V L S G A A N G K W F D K A C Graversen et al. 228 (1998) TNF164LC A V L S G A A N G K W L D K R C Graversen et al. 229 (1998) TND165AC A V L S G A A N G K W F A K R C Graversen et al. 230 (1998) TND165EC A V L S G A A N G K W F E K R C Graversen et al. 231 (2000) TND165NC A V L S G A A N G K W F N K R C Graversen et al. 232 (2000) rMBP I207VC V T I V D N G L W N D V S C Iobst et al. (1994) 233 I207LC V T I V D N G L W N D L S C Iobst et al. (1994) 234 I207AC V T I V D N G L W N D A S C Iobst et al. (1994) 235 I207EC V T I V D N G L W N D E S C Torgensen et al. 236 (1996) Region 4EC V T I V Y I K R E K D N G L W N D I S C Torgensen et al. 237 (1996)Region 4P C V T I V Y I K S P S D N G L W N D I S C Torgensen et al. 238(1996) 207VY C V T I V D N G L W N D V Y C Burrows et al.  239 (1997)β34 C A H V W T S G Q W N D V Y C Burrows et al.  240 (1997) h-esl Y94FC V E I F I K R E K D V G M W N D E R C Kogan et al. (1995) 241 Y94RC V E I R I K R E K D V G M W N D E R C Kogan et al. (1995) 242 Y94DC V E I D I K R E K D V G M W N D E R C Kogan et al. (1995) 243 Y94AC V E I A I K R E K D V G M W N D E R C Kogan et al. (1995) 244 Y94SC V E I S I K R E K D V G M W N D E R C Kogan et al. (1995) 245 E107DC V E I Y I K R E K D V G M W N D D R C Kogan et al. (1995) 246 E107AC V E I Y I K R E K D V G M W N D A R C Kogan et al. (1995) 247 E107NC V E I Y I K R E K D V G M W N D N R C Kogan et al. (1995) 248 E107KC V E I Y I K R E K D V G M W N D K R C Kogan et al. (1995) 249 E107QC V E I Y I K R E K D V G M W N D Q R C Kogan et al. (1995) 250 R97DC V E I Y I K D E K D V G M W N D E R C Revelle et al.  251 (1996) R97SC V E I Y I K S E K D V G M W N D E R C Revelle et al.  252 (1996) R97EC V E I Y I K E E K D V G M W N D E R C Revelle et al.  253 (1996) h-pslK96Q C V E I Y I Q S P S A P G M W N D E H C Revelle et al.  254 (1996)K96R C V E I Y I R S P S A P G M W N D E H C Revelle et al.  255 (1996)K96E C V E I Y I E S P S A P G M W N D E H C Revelle et al.  256 (1996)S97A C V E I Y I K A P S A P G M W N D E H C Revelle et al.  257 (1996)S97D C V E I Y I K D P S A P G M W N D E H C Revelle et al.  258 (1996)S97R C V E I Y I K R P S A P G M W N D E H C Revelle et al.  259 (1996)REK C V E I Y I K R E K A P G M W N D E H C Revelle et al.  260 (1996)S99D C V E I Y I K S P D A P G M W N D E H C Revelle et al.  261 (1996)CHL V191A C A H V W T S G Q W N D A Y C Burrows et al.  262 (1997) Y192AC A H V W T S G Q W N D V A C Burrows et al.  263 (1997)Other TN CTLD derivatives SEQ ID CTDL Mut. TN sequence (one letter code)Reference NO hTN TNR169A S G A A N G K W F D K R C A D QGraversen et al. 264 (1998) TNS85G C I S R G G T L G T P Q TJaquinod et al. 265 (1999) Notes: hTN: human tetranectin; rMBP: ratmannose binding protein, rSP-A: rat surfactant protein-A, hSP-A: humansurfactant protein-A, rSP-D: rat surfactant protein-D; h-esl: humane-selectin; h-psl: human p-selectin; MGR: macrophage galactose receptor;RHL: rat hepatic lectin, CHL: chicken hepatic lectin

Normally the model CTLD is defined by having a 3D structure thatconforms to the secondary-structure arrangement illustrated in FIG. 1characterized by the following main secondary structure elements:

-   -   five β-strands and two α-helices sequentially appearing in the        order β1, α1, α2, β2, β3, β4, and β5, the β-strands being        arranged in two anti-parallel β-sheets, one composed of β1 and        β5, the other composed of β2, β3 and β4,    -   at least two disulfide bridges, one connecting al and β5 and one        connecting β3 and the polypeptide segment connecting β4 and β5,    -   a loop region consisting of two polypeptide segments, loop        segment A (LSA) connecting β2 and β3 and comprising typically        15-70 or, less typically, 5-14 amino acid residues, and loop        segment B (LSB) connecting β3 and β4 and comprising typically        5-12 or less typically, 2-4 amino acid residues.

However, also a CTLD, for which no precise 3D structure is available,can be used as a model CTLD, such CTLD being defined by showing sequencesimilarity to a previously recognised member of the CTLD family asexpressed by an amino acid sequence identity of at least 22%, preferablyat least 25% and more preferably at least 30%, and by containing thecysteine residues necessary for establishing the conserved two-disulfidebridge topology (i.e. Cys_(I), Cys_(II), Cys_(III) and Cys_(IV)). Theloop region, consisting of the loop segments LSA and LSB, and itsflanking β-strand structural elements can then be identified byinspection of the sequence alignment with the collection of CTLDs shownin FIG. 1, which provides identification of the sequence locations ofthe β2- and β3-strands with the further corroboration provided bycomparison of these sequences with the four-residue consensus sequences,β2cseq and β3cseq, and the β4 strand segment located typically atpositions −6 to −2 and less typically at positions −5 to −2 relative tothe conserved Cys_(III) residue and with the characteristic residues atpositions −5 and −3 as elucidated from Table 1 and deducted above underBACKGROUND OF THE INVENTION.

The same considerations apply for determining whether in a model CTLDthe α-helices and β-strands and connecting segments are conserved tosuch a degree that the scaffold structure of the CTLD is substantiallymaintained.

It may be desirable that up to 10, preferably up to 4, and morepreferably 1 or 2, amino acid residues are substituted, deleted orinserted in the α-helices and/or β-strands and/or connecting segments ofthe model CTLD. In particular, changes of up to 4 residues may be madein the β-strands of the model CTLD as a consequence of the introductionof recognition sites for one or more restriction endonucleases in thenucleotide sequence encoding the CTLD to facilitate the excision of partor all of the loop region and the insertion of an altered amino acidsequence instead while the scaffold structure of the CTLD issubstantially maintained.

Of particular interest are proteins wherein the model CTLD is that of atetranectin. Well known tetranectins the CTLDs of which can be used asmodel CTLDs are human tetranectin and murine tetranectin. The proteinsaccording to the invention thus comprise variants of such model CTLDs.

The proteins according to the invention may comprise N-terminal and/orC-terminal extensions of the CTLD variant, and such extensions may forexample contain effector, enzyme, further binding and/or multimerisingfunctions. In particular, said extension may be the non-CTLD-portions ofa native C-type lectin-like protein or C-type lectin or a “soluble”variant thereof lacking a functional transmembrane domain.

The proteins according to the invention may also be multimers of amoiety comprising the CTLD variant, e.g. derivatives of the nativetetranectin trimer.

In a preferred aspect the present invention provides a combinatoriallibrary of proteins having the scaffold structure of C-type lectin-likedomains (CTLD), said proteins comprising variants of a model CTLDwherein the α-helices and β-strands are conserved to such a degree thatthe scaffold structure of the CTLD is substantially maintained, whilethe loop region or parts of the loop region of the CTLD is randomizedwith respect to amino acid sequence and/or number of amino acidresidues.

The proteins making up such a library comprise variants of model CTLDsdefined as for the above proteins according to the invention, and thevariants may include the changes stated for those proteins.

In particular, the combinatorial library according to the invention mayconsist of proteins wherein the model CTLD is that of a tetranectin,e.g. that of human tetranectin or that of murine tetranectin.

The combinatorial library according to the invention may consist ofproteins comprising N-terminal and/or C-terminal extensions of the CTLDvariant, and such extensions may for example contain effector, enzyme,further binding and/or multimerising functions. In particular, saidextensions may be the non-CTLD-portions of a native C-type lectin-likeprotein or C-type lectin or a “soluble” variant thereof lacking afunctional transmembrane domain.

The combinatorial library according to the invention may also consist ofproteins that are multimers of a moiety comprising the CTLD variant,e.g. derivatives of the native tetranectin trimer.

The present invention also provides derivatives of a native tetranectinwherein up to 10, preferably up to 4, and more preferably 1 or 2, aminoacid residues are substituted, deleted or inserted in the α-helicesand/or β-strands and/or connecting segments of its CTLD as well asnucleic acids encoding such derivatives. Specific derivatives appearfrom SEQ ID Nos: 02, 04, 09, 11, 13, 15, 29, 31, 36, and 38; and nucleicacids comprising nucleotide inserts encoding specific tetranectinderivatives appear from SEQ ID Nos: 12, 14, 35, and 37.

The invention comprises a method of constructing a tetranectinderivative adapted for the preparation of a combinatorial libraryaccording to the invention, wherein the nucleic acid encoding thetetranectin derivative has been modified to generate endonucleaserestriction sites within nucleic acid segments encoding β2, β3 or β4, orup to 30 nucleotides upstream or downstream in the sequence from anynucleotide which belongs to a nucleic acid segment encoding β2, β3 orβ4.

The invention also comprises the use of a nucleotide sequence encoding atetranectin, or a derivative thereof wherein the scaffold structure ofits CTLD is substantially maintained, for preparing a library ofnucleotide sequences encoding related proteins by randomising part orall of the nucleic acid sequence encoding the loop region of its CTLD.

Further, the present invention provides nucleic acid comprising anynucleotide sequence encoding a protein according to the invention.

In particular, the invention provides a library of nucleic acidsencoding proteins of a combinatorial library according to the invention,in which the members of the ensemble of nucleic acids, that collectivelyconstitute said library of nucleic acids, are able to be expressed in adisplay system, which provides for a logical, physical or chemical linkbetween entities displaying phenotypes representing properties of thedisplayed expression products and their corresponding genotypes.

In such a library the display system may be selected from

-   -   (I) a phage display system such as        -   (1) a filamentous phage fd in which the library of nucleic            acids is inserted into            -   (a) a phagemid vector,            -   (b) the viral genome of a phage            -   (c) purified viral nucleic acid in purified single- or                double-stranded form, or        -   (2) a phage lambda in which the library is inserted into            -   (a) purified phage lambda DNA, or            -   (b) the nucleic acid in lambda phage particles; or    -   (II) a viral display system in which the library of nucleic        acids is inserted into the viral nucleic acid of a eukaryotic        virus such as baculovirus; or    -   (III) a cell-based display system in which the library of        nucleic acids is inserted into, or adjoined to, a nucleic acid        carrier able to integrate either into the host genome or into an        extrachromosomal element able to maintain and express itself        within the cell and suitable for cell-surface display on the        surface of        -   (a) bacterial cells,        -   (b) yeast cells, or        -   (c) mammalian cells; or    -   (IV) a nucleic acid entity suitable for ribosome linked display        into which the library of nucleic acid is inserted; or    -   (V) a plasmid suitable for plasmid linked display into which the        library of nucleic acid is inserted.

A well-known and useful display system is the “Recombinant PhageAntibody System” with the phagemid vector “pCANTAB 5E” supplied byAmersham Pharmacia Biotech (code no. 27-9401-01).

Further, the present invention provides a method of preparing a proteinaccording to the invention, wherein the protein comprises at least oneor more, identical or not identical, CTLD domains with novel loop-regionsequences which has (have) been isolated from one or more CTLD librariesby screening or selection. At least one such CTLD domain may have beenfurther modified by mutagenesis; and the protein containing at least oneCTLD domain may have been assembled from two or more components bychemical or enzymatic coupling or crosslinking.

Also, the present invention provides a method of preparing acombinatorial library according to the invention comprising thefollowing steps:

-   -   1) inserting nucleic acid encoding a protein comprising a model        CTLD into a suitable vector,    -   2) if necessary, introducing restriction endonuclease        recognition sites by site directed mutagenesis, said recognition        sites being properly located in the sequence at or close to the        ends of the sequence encoding the loop region of the CTLD or        part thereof,    -   3) excising the DNA fragment encoding the loop region or part        thereof by use of the proper restriction endonucleases,    -   4) ligating mixtures of DNA fragments into the restricted        vector, and    -   5) inducing the vector to express randomized proteins having the        scaffold structure of CTLDs in a suitable medium.

In a further aspect, the present invention provides a method ofscreening a combinatorial library according to the invention for bindingto a specific target which comprises the following steps:

-   -   1) expressing a nucleic acids library to display the library of        proteins in the display system;    -   2) contacting the collection of entities displayed with a        suitably tagged target substance for which isolation of a        CTLD-derived exhibiting affinity for said target substance is        desired;    -   3) harvesting subpopulations of the entities displayed that        exhibit affinity for said target substance by means of        affinity-based selective extractions, utilizing the tag to which        said target substance is conjugated or physically attached or        adhering to as a vehicle or means of affinity purification, a        procedure commonly referred to in the field as “affinity        panning”, followed by re-amplification of the sub-library;    -   4) isolating progressively better binders by repeated rounds of        panning and re-amplification until a suitably small number of        good candidate binders is obtained; and,    -   5) if desired, isolating each of the good candidates as an        individual clone and subjecting it to ordinary functional and        structural characterisation in preparation for final selection        of one or more preferred product clones.

In a still further aspect, the present invention provides a method ofreformatting a protein according to the invention or selected from acombinatorial library according to the invention and containing a CTLDvariant exhibiting desired binding properties, in a desired alternativespecies-compatible framework by excising the nucleic acid fragmentencoding the loop region-substituting polypeptide and any requiredsingle framework mutations from the nucleic acid encoding said proteinusing PCR technology, site directed mutagenesis or restriction enzymedigestion and inserting said nucleic acid fragment into the appropriatelocation(s) in a display—or protein expression vector that harbours anucleic acid sequence encoding the desired alternative CTLD framework.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequences of ten CTLDs ofknown 3D-structure. The sequence locations of main secondary structureelements are indicated above each sequence, labelled in sequentialnumerical order as “αN”, denoting α-helix number N, and “βM”, denoting(β-strand number M.

The four cysteine residues involved in the formation of the twoconserved disulfide bridges of CTLDs are indicated and enumerated in theFigure as “C_(I)”, “C_(II)”, “C_(III)” and “C_(IV)”, respectively. Thetwo conserved disulfide bridges are C_(I)-C_(IV) and C_(II)-C_(III),respectively.

The ten C-type lectins are

-   hTN: human tetranectin [Nielsen et al. (1997)];-   MBP: mannose binding protein [Weis et al. (1991); Sheriff et al.    (1994)];-   SP-D: surfactant protein D [Håkansson et al. (1999)];-   LY49A: NK receptor LY49A [Tormo et al. (1999)];-   H1-ASR: H1 subunit of the asialoglycoprotein receptor [Meier et al.    (2000)];-   MMR-4: macrophage mannose receptor domain 4 [Feinberg et al.    (2000)];-   IX-A and IX-B: coagulation factors IX/X-binding protein domain A and    B, respectively [Mizuno et al. (1997)];-   Lit: lithostatine [Bertrand et al. (1996)];-   TU14: tunicate C-type lectin [Poget et al. (1999)].

FIG. 2 shows an alignment of the nucleotide and amino acid sequences ofthe coding regions of the mature forms of human and murine tetranectinwith an indication of known secondary structural elements.

-   hTN: human tetranectin; nucleotide sequence from Berglund and    Petersen (1992).-   mTN: murine tetranectin; nucleotide sequence from Sorensen et al.    (1995).-   Secondary structure elements from Nielsen et al. (1997).

“α” denotes an α-helix; “β” denotes a β-strand; and “L” denotes a loop.

FIG. 3 shows an alignment of the nucleotide and amino acid sequences ofhuman and murine tlec coding regions.

htlec: the sequence derived from hTN; mtlec: the sequence derived frommTN. The position of the restriction endonuclease sites for Bgl II, KpnI, and Mun I are indicated.

FIG. 4 shows an alignment of the nucleotide and amino acid sequences ofhuman and murine tCTLD coding regions. htCTLD: the sequence derived fromhTN; mtCTLD: the sequence derived from mTN. The position of therestriction endonuclease sites for Bgl II, Kpn I, and Mun I areindicated.

FIG. 5 shows an outline of the pT7H6FX-htlec expression plasmid. TheFX-htlec fragment was inserted into pT7H6 [Christensen et al. (1991)]between the Bam HI and Hind III cloning sites.

FIG. 6 shows the amino acid sequence (one letter code) of the FX-htlecpart of the H6FX-htlec fusion protein produced by pT7H6FX-htlec.

FIG. 7 shows an outline of the pT7H6FX-htCTLD expression plasmid. TheFX-htCTLD fragment was inserted into pT7H6 [Christensen et al. (1991)]between the Bam HI and Hind III cloning sites.

FIG. 8 shows the amino acid sequence (one letter code) of the FX-htCTLDpart of the H6FX-htCTLD fusion protein produced by pT7H6FX-htCTLD.

FIG. 9 shows an outline of the pPhTN phagemid. The PhTN fragment wasinserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech, codeno. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 10 shows the amino acid sequence (one letter code) of the PhTN partof the PhTN-gene III fusion protein produced by pPhTN.

FIG. 11 shows an outline of the pPhTN3 phagemid. The PhTN3 fragment wasinserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech, codeno. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 12 shows the amino acid sequence (one letter code) of the PhTN3part of the PhTN3-gene III fusion protein produced by pPhTN3.

FIG. 13 shows an outline of the pPhtlec phagemid. The Phtlec fragmentwas inserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech,code no. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 14 shows the amino acid sequence (one letter code) of the Phtlecpart of the Phtlec-gene III fusion protein produced by pPhtlec.

FIG. 15 shows an outline of the pPhtCTLD phagemid. The PhtCTLD fragmentwas inserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech,code no. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 16 shows the amino acid sequence (one letter code) of the PhtCTLDpart of the PhtCTLD-gene III fusion protein produced by pPhtCTLD.

FIG. 17 shows an outline of the pUC-mtlec.

FIG. 18 shows an outline of the pT7H6FX-mtlec expression plasmid. TheFX-mtlec fragment was inserted into pT7H6 [Christensen et al. (1991)]between the Bam HI and Hind III cloning sites.

FIG. 19 shows the amino acid sequence (one letter code) of the

FX-mtlec part of the H6FX-mtlec fusion protein produced bypT7H6FX-mtlec.

FIG. 20 shows an outline of the pT7H6FX-mtCTLD expression plasmid. TheFX-mtCTLD fragment was inserted into pT7H6 [Christensen et al. (1991)]between the Bam HI and Hind III cloning sites.

FIG. 21 shows the amino acid sequence (one letter code) of the FX-mtCTLDpart of the H6FX-mtCTLD fusion protein produced by pT7H6FX-mtCTLD.

FIG. 22 shows an outline of the pPmtlec phagemid. The Pmtlec fragmentwas inserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech,code no. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 23 shows the amino acid sequence (one letter code) of the Pmtlecpart of the Pmtlec-gene III fusion protein produced by pPmtlec.

FIG. 24 shows an outline of the pPmtCTLD phagemid. The PmtCTLD fragmentwas inserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech,code no. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 25 shows the amino acid sequence (one letter code) of the PmtCTLDpart of the PmtCTLD-gene III fusion protein produced by pPmtCTLD.

FIG. 26 shows an ELISA-type analysis of Phtlec-, PhTN3-, and M13K07helper phage binding to anti-tetranectin or BSA. Panel A: Analysis with3% skimmed milk/5 mM EDTA as blocking reagent. Panel B: Analysis with 3%skimmed milk as blocking reagent.

FIG. 27 shows an ELISA-type analysis of Phtlec-, PhTN3-, and M13K07helper phage binding to plasminogen (Plg) and BSA. Panel A: Analysiswith 3% skimmed milk/5 mM EDTA as blocking reagent. Panel B: Analysiswith 3% skimmed milk as blocking reagent.

FIG. 28 shows an ELISA-type analysis of the B series and C seriespolyclonal populations, from selection round 2, binding to plasminogen(Plg) compared to background.

FIG. 29 Phages from twelve clones isolated from the third round ofselection analyzed for binding to hen egg white lysozyme, humanβ₂-microglobulin and background in an ELISA-type assay.

FIG. 30 shows the amino acid sequence (one letter code) of the PrMBPpart of the PrMBP-gene III fusion protein produced by pPrMBP.

FIG. 31 shows an outline of the pPrMBP phagemid. The PrMBP fragment wasinserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech, codeno. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 32 shows the amino acid sequence (one letter code) of the PhSP-Dpart of the PhSP-D-gene III fusion protein produced by pPhSP-D.

FIG. 33 shows an outline of the pPhSP-D phagemid. The PhSP-D fragmentwas inserted into the phagemid pCANTAB 5E (Amersham Pharmacia Biotech,code no. 27-9401-01) between the Sfi I and Not I restriction sites.

FIG. 34. Phages from 48 clones isolated from the third round ofselection in the #1 series analyzed for binding to hen egg whitelysozyme and to A-HA in an ELISA-type assay.

FIG. 35. Phages from 48 clones isolated from the third round ofselection in the #4 series analyzed for binding to hen egg whitelysozyme and to A-HA in an ELISA-type assay.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms “C-type lectin-like protein” and “C-type lectin” are used torefer to any protein present in, or encoded in the genomes of, anyeukaryotic species, which protein contains one or more CTLDs or one ormore domains belonging to a subgroup of CTLDs, the CRDs, which bindcarbohydrate ligands. The definition specifically includes membraneattached C-type lectin-like proteins and C-type lectins, “soluble”C-type lectin-like proteins and C-type lectins lacking a functionaltransmembrane domain and variant C-type lectin-like proteins and C-typelectins in which one or more amino acid residues have been altered invivo by glycosylation or any other post-synthetic modification, as wellas any product that is obtained by chemical modification of C-typelectin-like proteins and C-type lectins.

In the claims and throughout the specification certain alterations maybe defined with reference to amino acid residue numbers of a CTLD domainor a CTLD-containing protein. The amino acid numbering starts at thefirst N-terminal amino acid of the CTLD or the native or artificialCTLD-containing protein product, as the case may be, which shall in eachcase be indicated by unambiguous external literature reference orinternal reference to a figure contained herein within the textualcontext.

The terms “amino acid”, “amino acids” and “amino acid residues” refer toall naturally occurring L-α-amino acids. This definition is meant toinclude norleucine, ornithine, and homocysteine. The amino acids areidentified by either the single-letter or three-letter designations:

Asp D aspartic acid Ile I isoleucine Thr T threonine Leu L leucine Ser Sserine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine Pro Pproline His H histidine Gly G glycine Lys K lysine Ala A alanine Arg Rarginine Cys C cysteine Trp W tryptophan Val V valine Gln Q glutamineMet M methionine Asn N asparagine Nle J norleucine Orn O ornithine Hcy Uhomocysteine Xxx X any L-α-amino acid.

The naturally occurring L-α-amino acids may be classified according tothe chemical composition and properties of their side chains. They arebroadly classified into two groups, charged and uncharged. Each of thesegroups is divided into subgroups to classify the amino acids moreaccurately:

-   -   A. Charged Amino Acids        -   Acidic Residues: Asp, Glu        -   Basic Residues: Lys, Arg, His, Orn    -   B. Uncharged Amino Acids        -   Hydrophilic Residues: Ser, Thr, Asn, Gln        -   Aliphatic Residues: Gly, Ala, Val, Leu, Ile, Nle        -   Non-polar Residues: Cys, Met, Pro, Hcy        -   Aromatic Residues: Phe, Tyr, Trp

The terms “amino acid alteration” and “alteration” refer to amino acidsubstitutions, deletions or insertions or any combinations thereof in aCTLD amino acid sequence. In the CTLD variants of the present inventionsuch alteration is at a site or sites of a CTLD amino acid sequence.Substitutional variants herein are those that have at least one aminoacid residue in a native CTLD sequence removed and a different aminoacid inserted in its place at the same position. The substitutions maybe single, where only one amino acid in the molecule has beensubstituted, or they may be multiple, where two or more amino acids havebeen substituted in the same molecule.

The designation of the substitution variants herein consists of a letterfollowed by a number followed by a letter. The first (leftmost) letterdesignates the amino acid in the native (unaltered) CTLD orCTLD-containing protein. The number refers to the amino acid positionwhere the amino acid substitution is being made, and the second(righthand) letter designates the amino acid that is used to replace thenative amino acid. As mentioned above, the numbering starts with “1”designating the N-terminal amino acid sequence of the CTLD or theCTLD-containing protein, as the case may be. Multiple alterations areseparated by a comma (,) in the notation for ease of reading them.

The terms “nucleic acid molecule encoding”, “DNA sequence encoding”, and“DNA encoding” refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide chain. The DNA sequence thus encodes the amino acidsequence.

The terms “mutationally randomized sequence”, “randomized polypeptidesegment”, “randomized amino acid sequence”, “randomized oligonucleotide”and “mutationally randomized sequence”, as well as any similar termsused in any context to refer to randomized sequences, polypeptides ornucleic acids, refer to ensembles of polypeptide or nucleic acidsequences or segments, in which the amino acid residue or nucleotide atone or more sequence positions may differ between different members ofthe ensemble of polypeptides or nucleic acids, such that the amino acidresidue or nucleotide occurring at each such sequence position maybelong to a set of amino acid residues or nucleotides that may includeall possible amino acid residues or nucleotides or any restricted subsetthereof. Said terms are often used to refer to ensembles in which thenumber of amino acid residues or nucleotides is the same for each memberof the ensemble, but may also be used to refer to such ensembles inwhich the number of amino acid residues or nucleotides in each member ofthe ensemble may be any integer number within an appropriate range ofinteger numbers.

II. Construction and Utility of Combinatorial CTLD Libraries

Several systems displaying phenotype, in terms of putative ligandbinding modules or modules with putative enzymatic activity, have beendescribed. These include: phage display (e.g. the filamentous phage fd[Dunn (1996), Griffiths amd Duncan (1998), Marks et al. (1992)], phagelambda [Mikawa et al. (1996)]), display on eukarotic virus (e.g.baculovirus [Ernst et al. (2000)]), cell display (e.g. display onbacterial cells [Benhar et al. (2000)], yeast cells [Boder and Wittrup(1997)], and mammalian cells [Whitehorn et al. (1995)], ribosome linkeddisplay [Schaffitzel et al. (1999)], and plasmid linked display [Gateset al. (1996)].

The most commonly used method for phenotype display and linking this togenotype is by phage display. This is accomplished by insertion of thereading frame encoding the scaffold protein or protein of interest intoan intra-domain segment of a surface exposed phage protein. Thefilamentous phage fd (e.g. M13) has proven most useful for this purpose.Polypeptides, protein domains, or proteins are the most frequentlyinserted either between the “export” signal and domain 1 of the fd geneIII protein or into a so-called hinge region between domain 2 and domain3 of the fd-phage gene III protein. Human antibodies are the mostfrequently used proteins for the isolation of new binding units, butother proteins and domains have also been used (e.g. human growthhormone [Bass et al. (1990)], alkaline phosphatase [McCafferty et al.(1991)], β-lactamase inhibitory protein [Huang et al. (2000)], andcytotoxic T lymphocyte-associated antigen 4 [Hufton et al. (2000)]. Theantibodies are often expressed and presented as scFv or Fab fusionproteins. Three strategies have been employed. Either a specificantibody is used as a scaffold for generating a library of mutationallyrandomized sequences within the antigen binding clefts [e.g. Fuji et al.(1998)] or libraries representing large ensembles of human antibodyencoding genes from non-immunised hosts [e.g. Nissim et al. (1994)] orfrom immunised hosts [e.g. Cyr and Hudspeth (2000)] are cloned into thefd phage vector.

The general procedure for accomplishing the generation of a displaysystem for the generation of CTLD libraries comprise essentially

-   -   (1) identification of the location of the loop-region, by        referring to the 3D structure of the CTLD of choice, if such        information is available, or, if not, identification of the        sequence locations of the β2-, β3- and β4 strands by sequence        alignment with the sequences shown in FIG. 1, as aided by the        further corroboration by identification of sequence elements        corresponding to the β2 and β3 consensus sequence elements and        β4-strand characteristics, also disclosed above;    -   (2) subcloning of a nucleic acid fragment encoding the CTLD of        choice in a protein display vector system with or without prior        insertion of endonuclease restriction sites close to the        sequences encoding β2, β3 and β4; and    -   (3) substituting the nucleic acid fragment encoding some or all        of the loop-region of the CTLD of choice with randomly selected        members of an ensemble consisting of a multitude of nucleic acid        fragments which after insertion into the nucleic acid context        encoding the receiving framework will substitute the nucleic        acid fragment encoding the original loop-region polypeptide        fragments with randomly selected nucleic acid fragments. Each of        the cloned nucleic acid fragments, encoding a new polypeptide        replacing an original loop-segment or the entire loop-region,        will be decoded in the reading frame determined within its new        sequence context.

Nucleic acid fragments may be inserted in specific locations intoreceiving nucleic acids by any common method of molecular cloning ofnucleic acids, such as by appropriately designed PCR manipulations inwhich chemically synthesized nucleic acids are copy-edited into thereceiving nucleic acid, in which case no endonuclease restriction sitesare required for insertion. Alternatively, the insertion/excision ofnucleic acid fragments may be facilitated by engineering appropriatecombinations of endonuclease restriction sites into the target nucleicacid into which suitably designed oligonucleotide fragments may beinserted using standard methods of molecular cloning of nucleic acids.

It will be apparent that interesting CTLD variants isolated from CTLDlibraries in which restriction endonuclease sites have been inserted forconvenience may contain mutated or additional amino acid residues thatneither correspond to residues present in the original CTLD nor areimportant for maintaining the interesting new affinity of the CTLDvariant. If desirable, e.g. in case the product needs to be rendered asnon-immunogenic as possible, such residues may be altered or removed byback-mutation or deletion in the specific clone, as appropriate.

The ensemble consisting of a multitude of nucleic acid fragments may beobtained by ordinary methods for chemical synthesis of nucleic acids bydirecting the step-wise synthesis to add pre-defined combinations ofpure nucleotide monomers or a mixture of any combination of nucleotidemonomers at each step in the chemical synthesis of the nucleic acidfragment. In this way it is possible to generate any level of sequencedegeneracy, from one unique nucleic acid sequence to the most complexmixture, which will represent a complete or incomplete representation ofmaximum number unique sequences of 4^(N), where N is the number ofnucleotides in the sequence.

Complex ensembles consisting of multitudes of nucleic acid fragmentsmay, alternatively, be prepared by generating mixtures of nucleic acidfragments by chemical, physical or enzymatic fragmentation ofhigh-molecular mass nucleic acid compositions like, e.g., genomicnucleic acids extracted from any organism. To render such mixtures ofnucleic acid fragments useful in the generation of molecular ensembles,as described here, the crude mixtures of fragments, obtained in theinitial cleavage step, would typically be size-fractionated to obtainfragments of an approximate molecular mass range which would thentypically be adjoined to a suitable pair of linker nucleic acids,designed to facilitate insertion of the linker-embedded mixtures ofsize-restricted oligonucleotide fragments into the receiving nucleicacid vector.

To facilitate the construction of combinatorial CTLD libraries intetranectin, the model CTLD of the preferred embodiment of theinvention, suitable restriction sites located in the vicinity of thenucleic acid sequences encoding β2, β3 and β4 in both human and murinetetranectin were designed with minimal perturbation of the polypeptidesequence encoded by the altered sequences. It was found possible toestablish a design strategy, as detailed below, by which identicalendonuclease restriction sites could be introduced at correspondinglocations in the two sequences, allowing interesting loop-regionvariants to be readily excised from a recombinant murine CTLD andinserted correctly into the CTLD framework of human tetranectin or viceversa.

Analysis of the nucleotide sequence encoding the mature form of humantetranectin reveals (FIG. 2) that a recognition site for the restrictionendonuclease Bgl II is found at position 326 to 331 (AGATCT), involvingthe encoded residues Glu109, Ile110, and Trp111 of β2, and that arecognition site for the restriction endonuclease Kas I is found atposition 382 to 387 (GGCGCC), involving the encoded amino acid residuesGly128 and Ala129 (located C-terminally in loop 2).

Mutation, by site directed mutagenesis, of G513 to A and of C514 to T inthe nucleotide sequence encoding human tetranectin would introduce a MunI restriction endonuclease recognition site therein, located at position511 to 516, and mutation of G513 to A in the nucleotide sequenceencoding murine tetranectin would introduce a Mun I restrictionendonuclease site therein at a position corresponding to the Mun I sitein human tetranectin, without affecting the amino acid sequence ofeither of the encoded protomers. Mutation, by site directed mutagenesis,of C327 to G and of G386 to C in the nucleotide sequence encoding murinetetranectin would introduce a Bgl II and a Kas I restrictionendonuclease recognition site, respectively, therein. Additionally, A325in the nucleotide sequence encoding murine tetranectin is mutagenized toa G. These three mutations would affect the encoded amino acid sequenceby substitution of Asn109 to Glu and Gly129 to Ala, respectively. Now,the restriction endonuclease Kas I is known to exhibit marked sitepreference and cleaves only slowly the tetranectin coding region.Therefore, a recognition site for another restriction endonucleasesubstituting the Kas I site is preferred (e.g. the recognition site forthe restriction endonuclease Kpn I, recognition sequence GGTACC). Thenucleotide and amino acid sequences of the resulting tetranectinderivatives, human tetranectin lectin (htlec) and murine tetranectinlectin (mtlec) are shown in FIG. 3. The nucleotide sequences encodingthe htlec and mtlec protomers may readily be subcloned into devicesenabling protein display of the linked nucleotide sequence (e.g.phagemid vectors) and into plasmids designed for heterologous expressionof protein [e.g. pT7H6, Christensen et al. (1991)]. Other derivativesencoding only the mutated CTLDs of either htlec or mtlec (htCTLD andmtCTLD, respectively) have also been constructed and subcloned intophagemid vectors and expression plasmids, and the nucleotide and aminoacid sequences of these CTLD derivatives are shown in FIG. 4.

The presence of a common set of recognition sites for the restrictionendonucleases Bgl II, Kas I or Kpn I, and Mun I in the ensemble oftetranectin and CTLD derivatives allows for the generation of proteinlibraries with randomized amino acid sequence in one or more of theloops and at single residue positions in β4 comprising the lectin ligandbinding region by ligation of randomized oligonucleotides into properlyrestricted phagemid vectors encoding htlec, mtlec, htCTLD, or mtCTLDderivatives.

After rounds of selection on specific targets (e.g. eukaryotic cells,virus, bacteria, specific proteins, polysaccharides, other polymers,organic compounds etc.) DNA may be isolated from the specific phages,and the nucleotide sequence of the segments encoding the ligand-bindingregion determined, excised from the phagemid DNA and transferred to theappropriate derivative expression vector for heterologous production ofthe desired product. Heterologous production in a prokaryote may bepreferred because an efficient protocol for the isolation and refoldingof tetranectin and derivatives has been reported (International PatentApplication Publication WO 94/18227 A2).

A particular advantage gained by implementing the technology of theinvention, using tetranectin as the scaffold structure, is that thestructures of the murine and human tetranectin scaffolds are almostidentical, allowing loop regions to be swapped freely between murine andhuman tetranectin derivatives with retention of functionality. Swappingof loop regions between the murine and the human framework is readilyaccomplished within the described system of tetranectin derivativevectors, and it is anticipated, that the system can be extended toinclude other species (e.g. rat, old and new world monkeys, dog, cattle,sheep, goat etc.) of relevance in medicine or veterinary medicine inview of the high level of homology between man and mouse sequences, evenat the genetic level. Extension of this strategy to include more speciesmay be rendered possible as and when tetranectin is eventually clonedand/or sequenced from such species.

Because the C-type lectin ligand-binding region represents a differenttopological unit compared to the antigen binding clefts of theantibodies, we envisage that the selected binding specificities will beof a different nature compared to the antibodies. Further, we envisagethat the tetranectin derivatives may have advantages compared toantibodies with respect to specificity in binding sugar moieties orpolysaccharides. The tetranectin derivatives may also be advantageous inselecting binding specificities against certain natural or syntheticorganic compounds.

Several CTLDs are known to bind calcium ions, and binding of otherligands is often either dependent on calcium (e.g. the collectin familyof C-type lectins, where the calcium ion bound in site 2 is directlyinvolved in binding the sugar ligand [Weis and Drickamer (1996)]) orsensitive to calcium (e.g. tetranectin, where binding of calciuminvolves more of the side chains known otherwise to be involved inplasminogen kringle 4 binding [Graversen et al. (1998)]). The calciumbinding sites characteristic of the C-type lectin-like protein familyare comprised by residues located in loop 1, loop 4 and β-strand 4 andare dependent on the presence of a proline residue (often interspacingloop 3 and loop 4 in the structure), which upon binding is foundinvariantly in the cis conformation. Moreover, binding of calcium isknown to enforce structural changes in the CTLD loop-region [Ng et al.(1998a,b)]. We therefore envisage, that binding to a specific targetligand by members of combinational libraries with preserved CTLD metalbinding sites may be modulated by addition or removal of divalent metalions (e.g. calcium ions) either because the metal ion may be directlyinvolved in binding, because it is a competitive ligand, or becausebinding of the metal ion enforces structural rearrangements within theputative binding site.

The trimeric nature of several members of the C-type lectin and C-typelectin-like protein family, including tetranectin, and the accompanyingavidity in binding may also be exploited in the creation of bindingunits with very high binding affinity.

As can be appreciated from the disclosure above, the present inventionhas a broad general scope and a wide area of application. Accordingly,the following examples, describing various embodiments thereof, areoffered by way of illustration only, not by way of limitation.

EXAMPLE 1

Construction of Tetranectin Derived E. coli Expression Plasmids andPhagemids

The expression plasmid pT7H6FX-htlec, encoding the FX-htlec (SEQ IDNO:01) part of full length H6FX-htlec fusion protein, was constructed bya series of four consecutive site-directed mutagenesis experimentsstarting from the expression plasmid pT7H6-rTN 123 [Holtet et al.(1997)] using the QuickChange™ Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) and performed as described by themanufacturer. Mismatching primer pairs introducing the desired mutationswere supplied by DNA Technology (Aarhus, Denmark). An outline of theresulting pT7H6FX-htlec expression plasmid is shown in FIG. 5, and thenucleotide sequence of the FX-htlec encoding insert is given as SEQ IDNO:01. The amino acid sequence of the FX-htlec part of the H6FX-htlecfusion protein is shown in FIG. 6 and given as SEQ ID NO:02.

The expression plasmid pT7H6FX-htCTLD, encoding the FX-htCTLD (SEQ IDNO: 03) part of the H6FX-htCTLD fusion protein, was constructed byamplification and subcloning into the plasmid pT7H6 (i.e. amplificationin a polymerase chain reaction using the expression plasmid pT7H6-htlecas template, and otherwise the primers, conditions, and subcloningprocedure described for the construction of the expression plasmidpT7H6TN3 [Holtet et al. (1997)]. An outline of the resultingpT7H6FX-htCTLD expression plasmid is shown in FIG. 7, and the nucleotidesequence of the FX-htCTLD encoding insert is given as SEQ ID NO:03. Theamino acid sequence of the FX-htCTLD part of the H6FX-htCTLD fusionprotein is shown in FIG. 8 and given as SEQ ID NO:04.

The phagemids, pPhTN and pPhTN3, were constructed by ligation of the SfiI and Not I restricted DNA fragments amplified from the expressionplasmids pT7H6-rTN 123 (with the oligonucleotide primers5-CGGCTGAGCGGCCCAGCCGGCCATGGCCGAGCCACCAACCCAGAAGC-3′ [SEQ ID NO:05] and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:06]) and pT7H6FX-htCTLD(with the oligonucleotide primers5′-CGGCTGAGCGGCCCAGCCGGCCATGGCCGCCCTGCAGACGGTC-3′ [SEQ ID NO:07] and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:06]), respectively, into aSfi I and Not I precut vector, pCANTAB 5E supplied by Amersham PharmaciaBiotech (code no. 27-9401-01) using standard procedures. Outlines of theresulting pPhTN and pPhTN3 phagemids are shown in FIG. 9 and FIG. 11,respectively, and the nucleotide sequences of the PhTN and PhTN3 insertsare given as SEQ ID NO:08 and SEQ ID NO:10, respectively. The amino acidsequences encoded by the PhTN and PhTN3 inserts are shown in FIG. 10(SEQ ID NO:09) and FIG. 12 (SEQ ID NO:11), respectively.

The phagemids, pPhtlec and pPhtCTLD, were constructed by ligation of theSfi I and Not I restricted DNA fragments amplified from the expressionplasmids pT7H6FX-htlec (with the oligonucleotide primers5-CGGCTGAGCGGCCCAGCCGGCCATGGCCGAGCCACCAACCCAGAAGC-3′ [SEQ ID NO:05] and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:06]) and pT7H6FX-htCTLD(with the oligonucleotide primers5′-CGGCTGAGCGGCCCAGCCGGCCATGGCCGCCCTGCAGACGGTC-3′ [SEQ ID NO:07] and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:06]), respectively, into aSfi I and Not I precut vector, pCANTAB 5E supplied by Amersham PharmaciaBiotech (code no. 27-9401-01) using standard procedures. Outlines of theresulting pPhtlec and pPhtCTLD phagemids are shown in FIG. 13 and FIG.15, respectively, and the nucleotide sequences of the Phtlec and PhtCTLDinserts are given as SEQ ID NO:12 and SEQ ID NO:14, repectively. Theamino acid sequences encoded by the Phtlec and PhtCTLD inserts are shownin FIG. 14 (SEQ ID NO:13) and FIG. 16 (SEQ ID NO:15), respectively.

A plasmid clone, pUC-mtlec, containing the nucleotide sequencecorresponding to the murine tetranectin derivative mtlec (FIG. 3 and SEQID NO:16) was constructed by four successive subclonings of DNAsubfragments in the following way: First, two oligonucleotides5′-CGGAATTCGAGTCACCCACTCCCAAGGCCAAGAAGGCTGCAAATGCCAAGAAAGATTTGGTGAGCTCAAAGATGTTC-3′(SEQ ID NO:17) and5′-GCGGATCCAGGCCTGCTTCTCCTTCAGCAGGGCCACCTCCTGGGCCAGGACATCCATCCTGTTCTTGAGCTCCTCGAACATCTTTGAGCTCACC-3′(SEQ ID NO:18) were annealed and after a filling in reaction cut withthe restriction endonucleases Eco RI (GAATTC) and Bam HI (GGATCC) andligated into Eco RI and Bam HI precut pUC18 plasmid DNA. Second, a pairof oligonucleotides5′-GCAGGCCTTACAGACTGTGTGCCTGAAGGGCACCAAGGTGAACTTGAAGTGCCTCCTGGCCTTCACCCAACCGAAGACCTTCCATGAGGCGAGCGAG-3′(SEQ ID NO:19) and5′-CCGCATGCTTCGAACAGCGCCTCGTTCTCTAGCTCTGACTGCGGGGTGCCCAGCGTGCCCCCTTGCGAGATGCAGTCCTCGCTCGCCTCATGG-3′(SEQ ID NO:20) was annealed and after a filling in reaction cut with therestriction endonucleases Stu I (AGGCCT) and Sph I (GCATGC) and ligatedinto the Stu I and Sph I precut plasmid resulting from the firstligation. Third, an oligonucleotide pair5′-GGTTCGAATACGCGCGCCACAGCGTGGGCAACGATGCGGAGATCTAAATGCTCCCAATTGC-3′ (SEQID NO:21) and5′-CCAAGCTTCACAATGGCAAACTGGCAGATGTAGGGCAATTGGGAGCATTTAGATC-3′ (SEQ IDNO: 22) was annealed and after a filling in reaction cut with therestriction endonucleases BstB I (TTCGAA) and Hind III (AAGCTT) andligated into the BstB I and Hind III precut plasmid resulting from thesecond ligation. Fourth, an oligonucleotide pair5′-CGGAGATCTGGCTGGGCCTCAACGACATGGCCGCGGAAGGCGCCTGGGTGGACATGACCGGTACCCTCCTGGCCTACAAGAACTGG-3′(SEQ ID NO:23) and5′-GGGCAATTGATCGCGGCATCGCTTGTCGAACCTCTTGCCGTTGGCTGCGCCAGACAGGGCGGCGCAGTTCTCGGCTTTGCCGCCGTCGGGTTGCGTCGTGATCTCCGTCTCCCAGTTCTTGTAGGCCAGG-3′(SEQ ID NO:24) was annealed and after a filling in reaction cut with therestriction endonucleases Bgl II (AGATCT) and Mun I (CAATTG) and ligatedinto the Bgl II and Mun I precut plasmid resulting from the thirdligation. An outline of the pUC-mtlec plasmid is shown in FIG. 17, andthe resulting nucleotide sequence of the Eco RI to Hind III insert isgiven as SEQ ID NO:16.

The expression plasmids pT7H6FX-mtlec and pT7H6FX-mtCTLD may beconstructed by ligation of the Bam HI and Hind III restricted DNAfragments, amplified from the pUC-mtlec plasmid with the oligonucleotideprimer pair 5-CTGGGATCCATCCAGGGTCGCGAGTCACCCACTCCCAAGG-3′ (SEQ ID NO:25)and 5′-CCGAAGCTTACACAATGGCAAACTGGC-3′ (SEQ ID NO:26), and with theoligonucleotide primer pair5′-CTGGGATCCATCCAGGGTCGCGCCTTACAGACTGTGGTC-3′ (SEQ ID NO:27), and5′-CCGAAGCTTACACAATGGCAAACTGGC-3′ (SEQ ID NO:26), respectively, into BamHI and Hind III precut pT7H6 vector using standard procedures. Anoutline of the expression plasmids pT7H6FX-mtlec and pT7H6FX-mtCTLD isshown in FIG. 18 and FIG. 20, respectively, and the nucleotide sequencesof the FX-mtlec and FX-mtCTLD inserts are given as SEQ ID NO:28 and SEQID NO:30, respectively. The amino acid sequences of the FX-mtlec andFX-mtCTLD parts of the fusion proteins H6FX-mtlec and H6FX-mtCTLD fusionproteins are shown in FIG. 19 (SEQ ID NO:29) and FIG. 21 (SEQ ID NO:31),respectively.

The phagemids pPmtlec and pPmtCTLD may be constructed by ligation of theSfi I and Not I restricted DNA fragments (amplified from the pUC-mtlecplasmid with the oligonucleotide primer pair5-CGGCTGAGCGGCCCAGCCGGCCATGGCCGAGTCACCCACTCCCAAGG-3′ [SEQ ID NO:32], and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:33] and with theoligonucleotide primers5′-CGGCTGAGCGGCCCAGCCGGCCATGGCCGCCTTACAGACTGTGGTC-3′ [SEQ ID NO:34] and5′-CCTGCGGCCGCCACGATCCCGAACTGG-3′ [SEQ ID NO:33], respectively) into aSfi I and Not I precut vector pCANTAB 5E supplied by Amersham PharmaciaBiotech (code no. 27-9401-01) using standard procedures. Outlines of thepPmtlec and pPmtCTLD plasmids are shown in FIG. 22 and FIG. 24,respectively, and the resulting nucleotide sequences of the Pmtlec andPmtCTLD inserts are given as SEQ ID NO:35 and SEQ ID NO:37, repectively.The amino acid sequences encoded by the Pmtlec and PmtCTLD inserts areshown in FIG. 23 (SEQ ID NO: 36) and FIG. 25 (SEQ ID NO: 38),respectively.

EXAMPLE 2

Demonstration of Successful Display of Phtlec and PhTN3 on Phages.

In order to verify that the Phtlec and PhTN3 Gene III fusion proteinscan indeed be displayed by the recombinant phage particles, thephagemids pPhtlec and pPhTN3 (described in Example 1) were transformedinto E. coli TG1 cells and recombinant phages produced upon infectionwith the helper phage M13K07. Recombinant phages were isolated byprecipitation with poly(ethylene glycol) (PEG 8000) and samples ofPhtlec and PhTN3 phage preparations as well as a sample of helper phagewere subjected to an ELISA-type sandwich assay, in which wells of aMaxisorb (Nunc) multiwell plate were first incubated with anti-humantetranectin or bovine serum albumin (BSA) and blocked in skimmed milk orskimmed milk/EDTA. Briefly, cultures of pPhtlec and pPhTN3 phagemidtransformed TG1 cells were grown at 37° C. in 2xTY-medium supplementedwith 20 glucose and 100 mg/L ampicillin until A₆₀₀ reached 0.5. By thenthe helper phage, M13KO7, was added to a concentration of 5×10⁹ pfu/mL.The cultures were incubated at 37° C. for another 30 min before cellswere harvested by centrifugation and resuspended in the same culturevolume of 2×TY medium supplemented with 50 mg/L kanamycin and 100 mg/Lampicillin and transferred to a fresh set of flasks and grown for 16hours at 25° C. Cells were removed by centrifugation and the phagesprecipitated from 20 mL culture supernatant by the addition of 6 mL ofice cold 200 PEG 8000, 2.5 M NaCl. After mixing the solution was left onice for one hour and centrifuged at 4° C. to isolate the precipitatedphages. Each phage pellet was resuspended in 1 mL of 10 mM tris-HCl pH8, 1 mM EDTA (TE) and incubated for 30 min before centrifugation. Thephage containing supernatant was transferred to a fresh tube. Along withthe preparation of phage samples, the wells of a Maxisorb plate wascoated overnight with (70 μL) rabbit anti-human tetranectin (apolyclonal antibody from DAKO A/S, code no. A0371) in a 1:2000 dilutionor with (70 μL) BSA (10 mg/mL). Upon coating, the wells were washedthree times with PBS (2.68 mM KCl, 1.47 mM KH₂PO₄, 137 mM NaCl, 8.10 mMNa₂HPO₄, pH 7.4) and blocked for one hour at 37° C. with 280 μL ofeither 3% skimmed milk in PBS, or 3% skimmed milk, 5 mM EDTA in PBS.Anti-tetranectin coated and BSA coated wells were then incubated withhuman Phtlec-, PhTN3-, or helper phage samples for 1 hour and thenwashed 3 times in PBS buffer supplemented with the appropriate blockingagent. Phages in the wells were detected after incubation withHRP-conjugated anti-phage conjugate (Amersham Pharmacia, code no.27-9421-01) followed by further washing. HRP activities were thenmeasured in a 96-well ELISA reader using a standard HRP chromogenicsubstrate assay.

Phtlec and PhTN3 phages produced strong responses (14 times background)in the assay, irrespective of the presence or absence of EDTA in theblocking agent, whereas helper phage produced no response abovebackground readings in either blocking agent. Only low binding to BSAwas observed (FIG. 26).

It can therefore be concluded that the human Phtlec and PhTN3 phagesboth display epitopes that are specifically recognized by the anti-humantetranectin antibody.

EXAMPLE 3

Demonstration of authentic ligand binding properties of Phtlec and PhTN3displayed on phage

The apo-form of the CTLD domain of human tetranectin binds in alysine-sensitive manner specifically to the kringle 4 domain of humanplasminogen [Graversen et al. (1998)]. Binding of tetranectin toplasminogen can be inhibited by calcium which binds to two sites in theligand-binding site in the CTLD domain (Kd approx. 0.2 millimolar) or bylysine-analogues like AMCHA (6-amino-cyclohexanoic acid), which bindspecifically in the two stronger lysine-binding sites in plasminogen ofwhich one is located in kringle 1 and one is located in kringle 4 (Kdapprox. 15 micromolar).

To demonstrate specific AMCHA-sensitive binding of human Phtlec andPhTN3 phages to human plasminogen, an ELISA assay, in outline similar tothat employed to demonstrate the presence of displayed Phlec and PhCTLDGIII fusion proteins on the phage particles (cf. Example 2), wasdevised.

Wells were coated with solutions of human plasminogen (10 μg/mL), withor without addition of 5 mM AMCHA. Control wells were coated with BSA.Two identical arrays were established, one was subjected to blocking ofexcess binding capacity with 3% skimmed milk, and one was blocked using3% skimmed milk supplemented with 5 mM EDTA. Where appropriate,blocking, washing and phage stock solutions were supplemented by 5 mMAMCHA. The two arrays of wells were incubated with either Phtlec-, orPhTN3-, or helper phage samples, and after washing the amount of phagebound in each well was measured using the HRP-conjugated antiphageantibody as above. The results are shown in FIG. 27, panels A and B, andcan be summarized as follows

-   -   (a) In the absence of AMCHA, binding of human Phtlec phages to        plasminogen-coated wells generated responses at 8-10 times        background levels using either formulation of blocking agent,        whereas human PhTN3 phages generated responses at 4 (absence of        EDTA) or 7 (presence of EDTA) times background response levels.    -   (b) In the presence of 5 mM AMCHA, binding of human Phtlec- and        PhTN3 phages to plasminogen was found to be completely        abolished.    -   (c) Phtlec and PhTN3 phages showed no binding to BSA, and        control helper phages showed no binding to any of the        immobilized substances.    -   (d) Specific binding of human Phtlec and PhTN3 phages to a        specific ligand at moderate binding strength (about 20        micromolar level) can be detected with high efficiency at        virtually no background using a skimmed-milk blocking agent,        well-known in the art of combinatorial phage technology as a        preferred agent effecting the reduction of non-specific binding.

In conclusion, the results show that the Phtlec and PhTN3 Gene IIIfusion proteins displayed on the phage particles exhibitplasminogen-binding properties corresponding to those of authentictetranectin, and that the physical and biochemical properties of Phtlecand PhTN3 phages are compatible with their proposed use as vehicles forthe generation of combinatorial libraries from which CTLD derived unitswith new binding properties can be selected.

EXAMPLE 4

Construction of the phage libraries Phtlec-lb001 and Phtlec-lb002.

All oligonucleotides used in this example were supplied by DNATechnology (Aarhus, Denmark).

The phage library Phtlec-lb001, containing random amino acid residuescorresponding to Phtlec (SEQ ID NO: 12) positions 141-146 (loop 3),150-153 (part of loop 4), and residue 168 (Phe in β4), was constructedby ligation of 20 μg KpnI and MunI restricted pPhtlec phagemid DNA (cf,Example 1) with 10 μg of KpnI and MunI restricted DNA fragment amplifiedfrom the oligonucleotide htlec-lib1-tp (SEQ ID NO: 39), where N denotesa mixture of 25% of each of the nucleotides T, C, G, and A, respectivelyand S denotes a mixture of 50% of C and G, encoding the appropriatelyrandomized nucleotide sequence and the oligonucleotides htlec-lib1-rev(SEQ ID NO: 40) and htlec-lib1/2-fo (SEQ ID NO: 41) as primers usingstandard conditions. The ligation mixture was used to transformso-called electrocompetent E. coli TG-1 cells by electroporation usingstandard procedures. After transformation the E. coli TG-1 cells wereplated on 2×TY-agar plates containing 0.2 mg ampicillin/mL and 20glucose and incubated over night at 30° C.

The phage library Phtlec-lb002, containing random amino acid residuescorresponding to Phtlec (SEQ ID NO: 12) positions 121-123, 125 and 126(most of loop 1), and residues 150-153 (part of loop 4) was constructedby ligation of 20 μg BglII and MunI restricted pPhtlec phagemid DNA (cf,EXAMPLE 1) with 15 μg of BglII and MunI restricted DNA fragmentamplified from the pair of oligonucleotides htlec-lib2-tprev (SEQ ID NO:42) and htlec-lib2-tpfo (SEQ ID NO: 43), where N denotes a mixture of25% of each of the nucleotides T, C, G, and A, respectively and Sdenotes a mixture of 50% of C and G, encoding the appropriatelyrandomized nucleotide sequence and the oligonucleotides htlec-lib2-rev(SEQ ID NO: 44) and htlec-lib1/2-fo (SEQ ID NO: 41) as primers usingstandard conditions. The ligation mixture was used to transformso-called electrocompetent E. coli TG-1 cells by electroporation usingstandard procedures. After transformation the E. coli TG-1 cells wereplated on 2×TY-agar plates containing 0.2 mg ampicillin/mL and 20glucose and incubated overnight at 30° C.

The titer of the libraries Phtlec-lb001 and −lb002 was determined to1.4*10⁹ and 3.2*10⁹ clones, respectively. Six clones from each librarywere grown and phagemid DNA isolated using a standard miniprepprocedure, and the nucleotide sequence of the loop-region determined(DNA Technology, Aarhus, Denmark). One clone from each library failed,for technical reasons, to give reliable nucleotide sequence, and oneclone from Phtlec-lib001 apparently contained a major deletion. Thevariation of nucleotide sequences, compared to Phtlec (SEQ ID NO: 12),of the loop-regions of the other nine clones (lb001-1, lb001-2, lb001-3,lb001-4, lb002-1, lb002-2, lb002-3, lb002-4, and lb002-5) is shown inTable 3.

TABLE 3 Variation of Phtlec loop derivatives isolated from the librariesPhtlec-lb001 and - lb002. (β2 and β3 consensus elements are indicated)

EXAMPLE 5

Construction of the Phage Library PhtCTLD-lb003

All oligonucleotides used in this example were supplied by DNATechnology (Aarhus, Denmark).

The phage library PhtCTLD-lb003, containing random amino acid residuescorresponding to PhtCTLD (SEQ ID NO: 15) positions 77 to 79 and 81 to 82(loop 1) and 108 to 109 (loop 4) was constructed by ligation of 20 μgBglII and MunI restricted pPhtCTLD phagemid DNA (cf. Example 1) with 10g of a BglII and MunI restricted DNA fragment population encoding theappropriately randomized loop 1 and 4 regions with or without two andthree random residue insertions in loop 1 and with three and four randomresidue insertions in loop 4. The DNA fragment population was amplified,from six so-called assembly reactions combining each of the three loop 1DNA fragments with each of the two loop 4 DNA fragments as templates andthe oligonucleotides TN-lib3-rev (SEQ ID NO: 45) and loop 3-4-5 tagfo(SEQ ID NO: 46) as primers using standard procedures. Each of the threeloop 1 fragments was amplified in a reaction with either theoligonucleotides loop1b (SEQ ID NO: 47), loop1c (SEQ ID NO: 48), orloop1d (SEQ ID NO: 49) as template and the oligonucleotides TN-lib3-rev(SEQ ID NO: 45) and TN-KpnI-fo (SEQ ID NO: 50) as primers, and each ofthe two DNA loop 4 fragments was amplified in a reaction with either theoligonucleotide loop4b (SEQ ID NO: 51) or loop4c (SEQ ID NO: 52) astemplate and the oligonucleotides loop3-4rev (SEQ ID NO: 53) andloop3-4fo (SEQ ID NO: 54) as primers using standard procedures. In theoligonucleotide sequences N denotes a mixture of 25% of each of thenucleotides T, C, G, and A, respectively and S denotes a mixture of 50%of C and G, encoding the appropriately randomized nucleotide sequence.The ligation mixture was used to transform so-called electrocompetent E.coli TG-1 cells by electroporation using standard procedures. Aftertransformation the E. coli TG-1 cells were plated on 2×TY-agar platescontaining 0.2 mg ampicillin/mL and 20 glucose and incubated over nightat 30° C.

The size of the resulting library, PhtCTLD-lb003, was determined to1.4*10¹⁰ clones. Twenty four clones from the library were grown andphages and phagemid DNA isolated. The nucleotide sequences of theloop-regions were determined (DNA Technology, Aarhus, Denmark) andbinding to a polyclonal antibody against tetranectin, anti-TN (DAKO A/S,Denmark), analyzed in an ELISA-type assay using HRP conjugated anti-geneVIII (Amersham Pharmacia Biotech) as secondary antibody using standardprocedures. Eighteen clones were found to contain correct loop inserts,one clone contained the wild type loop region sequence, one a majordeletion, two contained two or more sequences, and two clones containeda frameshift mutation in the region. Thirteen of the 18 clones withcorrect loop inserts, the wild type clone, and one of the mixed isolatesreacted strongly with the polyclonal anti-TN antibody. Three of the 18correct clones reacted weakly with the antibody, whereas, two of thecorrect clones, the deletion mutant, one of the mixed, and the twoframeshift mutants did not show a signal above background.

EXAMPLE 6

Phage Selection by Biopanning on Anti-TN Antibody.

Approximately 10¹¹ phages from the PhtCTLD-lb003 library was used forselection in two rounds on the polyclonal anti-TN antibody by panning inMaxisorb immunotubes (NUNC, Denmark) using standard procedures. Fifteenclones out of 7*10⁷ from the plating after the second selection roundwere grown and phagemid DNA isolated and the nucleotide sequencedetermined. All 15 clones were found to encode correct and differentloop sequences.

EXAMPLE 7

Model Selection of CTLD-Phages on Plasminogen.

I: Elution by Trypsin Digestion After Panning.

In order to demonstrate that tetranectin derived CTLD bearing phages canbe selected from a population of phages, mixtures of PhtCTLD phagesisolated from a E. coli TG1 culture transformed with the phagemidpPhtCTLD (cf, EXAMPLE 1) after infection with M13K07 helper phage andphages isolated from a culture transformed with the phagemid pPhtCPBafter infection with M13K07 helper phage at ratios of 1:10 and 1:10⁵,respectively were used in a selection experiment using panning in96-well Maxisorb micro-titerplates (NUNC, Denmark) and with humanplasminogen as antigen. The pPhtCPB phagemid was constructed by ligationof the double stranded oligonucleotide (SEQ ID NO: 55) with theappropriate restriction enzyme overhang sequences into KpnI and MunIrestricted pPhtCTLD phagemid DNA. The pPhtCBP phages derived uponinfection with the helper phages displays only the wild type M13 geneIII protein because of the translation termination codons introducedinto the CTLD coding region of the resulting pPhtCPB phagemid (SEQ IDNO: 56).

The selection experiments were performed in 96 well micro titer platesusing standard procedures. Briefly, in each well 3 μg of humanplasminogen in 100 μL PBS (PBS, 0.2 g KCl, 0.2 g KH₂PO₄, 8 g NaCl, 1.44g Na₂HPO₄, 2H₂O, water to 1 L, and adjusted to pH 7.4 with NaOH) or 100μL PBS (for analysis of non specific binding) was used for over nightcoating at 4° C. and at 37° C. for one hour. After washing once withPBS, wells were blocked with 400 μL PBS and 3% non fat dried milk forone hour at 37° C. After blocking wells were washed once in PBS and 0.1%Tween 20 and three times with PBS before the addition of phagessuspended in 100 μL PBS, 3% non fat dried milk. The phages were allowedto bind at 37° C. for one hour before washing three times with PBS,Tween 20 and three times with PBS. Bound phages were eluted from eachwell by trypsin digestion in 100 μL (1 mg/mL trypsin in PBS) for 30 min.at room temperature, and used for infection of exponentially growing E.coli TG1 cells before plating and titration on 2×TY agar platescontaining 20 glucose and 0.1 mg/mL ampicillin.

Initially (round 1), 10¹² PhtCTLD phages (A series), a mixture of 10¹⁰PhtCTLD phages and 10¹¹ PhtCPB phages (B series), or a mixture of 10⁶PhtCTLD and 10¹¹ PhtCPB phages (C series) were used. In the followinground (round 2) 10¹¹ phages of the output from each series were used.Results from the two rounds of selection are summarised in Table 4.

TABLE 4 Selection of mixtures of PhtCTLD and PhtCPB by panning andelution with trypsin. Plasminogen Blank (*10⁵ colonies) (*10⁵ colonies)Round 1 A 113.0 19.50 B 1.8 1.10 C 0.1 0.30 Round 2 A 49 0.10 B 5.2 0.20C 0.3 0.04

Phagemid DNA from 12 colonies from the second round of plating togetherwith 5 colonies from a plating of the initial phage mixtures wasisolated and the nucleotide sequence of the CTLD region determined. Fromthe initial 1/10 mixture (B series) of PhtCTLD/PhtCPB one out of fivewere identified as the CTLD sequence. From the initial 1/10⁵ mixture (Cseries) all five sequences were derived from the pPhtCPB phagemid. Afterround 2 nine of the twelve sequences analyzed from the B series and alltwelve sequences from the C series were derived from the pPhtCTLDphagemid.

EXAMPLE 8

Model Selection of CTLD-Phages on Plasminogen.

II: Elution by 0.1 M Triethylamine After Panning.

In order to demonstrate that tetranectin derived CTLD-bearing phages canbe selected from a population of phages, mixtures of PhtCTLD phagesisolated from a E. coli TG1 culture transformed with the phagemidpPhtCTLD (cf, EXAMPLE 1) after infection with M13K07 helper phage andphages isolated from a culture transformed with the phagemid pPhtCPB(cf, EXAMPLE 6) after infection with M13K07 helper phage at ratios of1:10² and 1:10⁶, respectively were used in a selection experiment usingpanning in 96-well Maxisorb micro-titerplates (NUNC, Denmark) and withhuman plasminogen as antigen using standard procedures.

Briefly, in each well 3 μg of human plasminogen in 100 μL PBS (PBS, 0.2g KCl, 0.2 g KH₂PO₄, 8 g NaCl, 1.44 g Na₂HPO₄, 2H₂O, water to 1 L, andadjusted to pH 7.4 with NaOH) or 100 μL PBS (for analysis of nonspecific binding) was used for over night coating at 4° C. and at 37° C.for one hour. After washing once with PBS, wells were blocked with 400 LPBS and 3% non fat dried milk for one hour at 37° C. After blockingwells were washed once in PBS and 0.1% Tween 20 and three times with PBSbefore the addition of phages suspended in 100 μL PBS, 3% non fat driedmilk. The phages were allowed to bind at 37° C. for one hour beforewashing 15 times with PBS, Tween 20, and 15 times with PBS. Bound phageswere eluted from each well by 100 μL 0.1 M triethylamine for 10 min atroom temperature, and upon neutralisation with 0.5 vol. 1 M Tris-HCl pH7.4, used for infection of exponentially growing E. coli TG1 cellsbefore plating and titration on 2×TY agar plates containing 20 glucoseand 0.1 mg/mL ampicillin.

Initially (round 1) 10¹² PhtCTLD phages (A series), a mixture of 10⁹PhtCTLD phages and 10¹¹ PhtCPB phages (B series), or a mixture of 10⁵PhtCTLD and 10¹¹ PhtCPB phages (C series) were used. In the followinground (round 2) 10¹¹ phages of the output from each series were used.Results from the two rounds of selection are summarised in Table 5.

TABLE 5 Selection of mixtures of PhtCTLD and PhtCPB by panning elutionwith triethylamine. Plasminogen Blank (*10⁴ colonies) (*10⁴ colonies)Round 1 A 18 0.02 B 0.5 0.00 C 0.25 0.02 Round 2 A n.d. n.d. B 5.0 0.00C 1.8 0.02 Round 3 A n.d. n.d. B 11 0.00 C 6.5 0.02 n.d. = notdetermined

Phage mixtures from the A and the B series from the second round ofselection were grown using a standard procedure, and analyzed forbinding to plasminogen in an ELISA-type assay. Briefly, in each well 3μg of plasminogen in 100 μL PBS (PBS, 0.2 g KCl, 0.2 g KH2PO4, 8 g NaCl,1.44 g Na₂HPO₄, 2H₂O, water to 1 L, and adjusted to pH 7.4 with NaOH) or100 μL PBS (for analysis of non specific binding) was used for overnight coating at 4° C. and at 37° C. for one hour. After washing oncewith PBS, wells were blocked with 400 μL PBS and 3% non fat dried milkfor one hour at 370C. After blocking wells were washed once in PBS and0.1% Tween 20 and three times with PBS before the addition of phagessuspended in 100 μL PBS, 3% non fat dried milk. The phage mixtures wereallowed to bind at 37° C. for one hour before washing three times withPBS, Tween 20, and three times with PBS. After washing, 50 μL of a1:5000 dilution of a HRP-conjugated anti-gene VIII antibody (AmershamPharmacia Biotech) in PBS, 3% non fat dried milk was added to each welland incubated at 37° C. for one hour. After binding of the “secondary”antibody wells were washed three times with PBS, Tween 20, and threetimes with PBS before the addition of 50 μL of TMB substrate (DAKO-TMBOne-Step Substrate System, code: 51600, DAKO, Denmark). Reaction wasallowed to proceed for 20 min. before quenching with 0.5 vol. 0.5 MH₂SO₄, and analysis. The result of the ELISA analysis confirmed specificbinding to plasminogen of phages in both series (FIG. 28).

EXAMPLE 9

Selection of Phages From the Library Phtlec-lb002 Binding to Hen EggWhite Lysozyme.

1.2*10¹² phages, approximately 250 times the size of the originallibrary, derived from the Phtlec-lb002 library (cf, EXAMPLE 4) were usedin an experimental procedure for the selection of phages binding to henegg white lysozyme involving sequential rounds of panning using standardprocedures.

Briefly, 30 μg of hen egg white lysozyme in 1 mL PBS (PBS, 0.2 g KCl,0.2 g KH₂PO₄, 8 g NaCl, 1.44 g Na₂HPO₄, 2H₂O, water to 1 L, and adjustedto pH 7.4 with NaOH) or 1 mL PBS (for analysis of non specific binding)was used for over night coating of Maxisorb immunotubes (NUNC, Denmark)at 4° C. and at 37° C. for one hour. After washing once with PBS, tubeswere filled and blocked with PBS and 3% non fat dried milk for one hourat 37° C. After blocking tubes were washed once in PBS, 0.1% Tween 20and three times with PBS before the addition of phages suspended in 1 mLPBS, 3% non fat dried milk. The phages were allowed to bind at 37° C.for one hour before washing six times with PBS, Tween 20 and six timeswith PBS. Bound phages were eluted from each well by 1 mL 0.1 Mtriethylamine for 10 min at room temperature, and upon neutralisationwith 1 M Tris-HCl pH 7.4, used for infection of exponentially growing E.coli TG1 cells before plating and titration on 2×TY agar platescontaining 20 glucose and 0.1 mg/mL ampicillin. In the subsequent roundsof selection approximately 10¹² phages derived from a culture grown fromthe colonies plated after infection with the phages eluted from thelysozyme coated tube were used in the panning procedure. However, thestringency in binding was increased by increasing the number of washingstep after phage panning from six to ten.

The results from the selection procedure is shown in Table 7.

TABLE 7 Selection by panning of lysozyme binding phages fromPhtlec-lb002 library. Lysozyme Blank Ratio Round 1 2.4 * 10⁴ n.a. n.a.Round 2 3.5 * 10³ 4.0 * 10² 9 Round 3 3.2 * 10⁵ 2.5 * 10² 1.3 * 10³ n.a.= not applicable

Phages were grown from twelve clones isolated from the third round ofselection in order to analyse the specificity of binding using astandard procedure, and analyzed for binding to hen egg white lysozymeand human β₂-microglobulin in an ELISA-type assay. Briefly, in each well3 μg of hen egg white lysozyme in 100 μL PBS (PBS, 0.2 g KCl, 0.2 gKH₂PO₄, 8 g NaCl, 1.44 g Na₂HPO₄, 2H₂O, water to 1 L, and adjusted to pH7.4 with NaOH), or 3 μg of human β₂-microglobulin, or 100 μL PBS (foranalysis of non specific binding) was used for over night coating at 4°C. and at 37° C. for one hour. After washing once with PBS, wells wereblocked with 400 μL PBS and 3% non fat dried milk for one hour at 37° C.After blocking wells were washed once in PBS and 0.1% Tween 20 and threetimes with PBS before the addition of phages suspended in 100 μL PBS, 3%non fat dried milk. The phages were allowed to bind at 37° C. for onehour before washing three times with PBS, Tween 20 and three times withPBS. After washing, 50 μL of a 1 to 5000 dilution of a HRP-conjugatedanti-gene VIII antibody (Amersham Pharmacia Biotech) in PBS, 3% non fatdried milk was added to each well and incubated at 37° C. for one hour.After binding of the “secondary” antibody wells were washed three timeswith PBS, Tween 20 and three times with PBS before the addition of 50 μLof TMB substrate (DAKO-TMB One-Step Substrate System, code: 51600, DAKO,Denmark). Reaction was allowed to proceed for 20 min before quenchingwith 0.5 M H₂SO₄.

Results showing relatively weak but specific binding to lysozyme aresummarised in FIG. 29.

EXAMPLE 10

Construction of the Rat Mannose-Binding Protein CTLD (r-MBP) DerivedPhagemid (pPrMBP) and Human Lung Surfactant Protein D CTLD (h-SP-D)Derived Phagemid (pPhSP-D)

The phagemid, pPrMBP, is constructed by ligation of the Sfi I and Not Irestricted DNA fragment amplified from cDNA, isolated from rat liver(Drickamer, K., et al., J. Biol. Chem. 1987, 262(6):2582-2589) (with theoligonucleotide primers SfiMBP5′-CGGCTGAGCGGCCCAGCCGGCCATGGCCGAGCCAAACAAGTTGCATGCCTTCTCC-3′ [SEQ IDNO:62] and NotMBP 5′-GCACTCCTGCGGCCGCGGCTGGGAACTCGCAGAC-3′ [SEQ IDNO:63]) into a Sfi I and Not I precut vector, pCANTAB 5E supplied byAmersham Pharmacia Biotech (code no. 27-9401-01) using standardprocedures. Outlines of the resulting pPrMBP is shown in FIG. 31 and thenucleotide sequence of PrMBP is given as (SEQ ID NO:58). The amino acidsequence encoded by the PrMBP insert is shown in FIG. 30 (SEQ ID NO:59).

The phagemid,pPhSP-D, is constructed by ligation of the Sfi I and Not Irestricted DNA fragment amplified from cDNA, isolated from human lung(Lu, J., et al., Biochem J. 1992 Jun. 15; 284:795-802) (with theoligonucleotide primers SfiSP-D5′-CGGCTGAGCGGCCCAGCCGGCCATGGCCGAGCCAAAGAAAGTTGAGCTCTTCCC-3′ [SEQ IDNO:64] and NotSP-D 5′-GCACTCCTGCGGCCGCGAACTCGCAGACCACAAGAC-3′ [SEQ IDNO:65]) into a Sfi I and Not I precut vector, pCANTAB 5E supplied byAmersham Pharmacia Biotech (code no. 27-9401-01) using standardprocedures. Outlines of the resulting pPhSP-D is shown in FIG. 33 andthe nucleotide sequence of PhSP-D, is given as (SEQ ID NO:60). The aminoacid sequences encoded by the PhSP-D insert is shown in FIG. 32 (SEQ IDNO:61).

EXAMPLE 11

Construction of the Phage Library PrMBP-lb001

The phage library PrMBP-lb001, containing random amino acid residuescorresponding to PrMBP CTLD (SEQ ID NO:59) positions 71 to 73 or 70 to76 (loop 1) and 97 to 101 or 100 to 101 (loop 4) is constructed byligation of 20 μg SfiI and NotI restricted pPrMBP phagemid DNA (cf.Example 10) with 10 μg of a SfiI and NotI restricted DNA fragmentpopulation encoding the appropriately randomized loop 1 and 4 regions.The DNA fragment population is amplified, from nine assembly reactionscombining each of the three loop 1 DNA fragments with each of the threeloop 4 DNA fragments as templates and the oligonucleotides Sfi-tag5′-CGGCTGAGCGGCCCAGC-3′ (SEQ ID NO:74) and Not-tag5′-GCACTCCTGCGGCCGCG-3′ (SEQ ID NO:75) as primers using standardprocedures. Each of the three loop 1 fragments is amplified in a primaryPCR reaction with pPrMBP phagmid DNA (cf. Example 10) as template andthe oligonucleotides MBPloop1a fo (SEQ ID NO:66), MBPloop1b fo (SEQ IDNO:67)or MBPloop1c fo (SEQ ID NO:68) and SfiMBP (SEQ ID NO:62) asprimers, and further amplified in a secondary PCR reaction using Sfi-tag(SEQ ID NO:74) and MBPloop1-tag fo (SEQ ID NO:69). Each of the three DNAloop 4 fragments is amplified in a primary PCR reaction with pPrMBPphagemid DNA (cf. Example 10) as template and the oligonucleotidesMBPloop4a rev (SEQ ID NO:71), MBPloop4b rev (SEQ ID NO:72) or MBPloop4crev (SEQ ID NO:73) and NotMBP (SEQ ID NO:63) as primers using standardprocedures and further amplified in a secondary PCR reaction usingMBPloop4-tag rev (SEQ ID NO:70) and Not-tag (SEQ ID NO:63). In theoligonucleotide sequences N denotes a mixture of 25% of each of thenucleotides T, C, G, and A, respectively, and S denotes a mixture of 50%of C and G, encoding the appropriately randomized nucleotide sequence.The ligation mixture is used to transform so-called electrocompetent E.coli TG-1 cells by electroporation using standard procedures. Aftertransformation the E. coli TG-1 cells are plated on 2×TY-agar platescontaining 0.2 mg ampicillin/mL and 20 glucose and incubated over nightat 30° C.

EXAMPLE 12

Construction of the Phage Library PhSP-D-lb001

The phage library PhSP-D-lb001, containing random amino acid residuescorresponding to PhSP-D CTLD insert (SEQ ID NO:61) positions 74 to 76 or73 to 79 (loop 1) and 100 to 104 or 103 to 104 (loop 4) is constructedby ligation of 20 μg SfiI and NotI restricted pPhSP-D phagemid DNA (cf.Example 10) with 10 of a SfiI and NotI restricted DNA fragmentpopulation encoding the appropriately randomized loop 1 and 4 regions.The DNA fragment population is amplified, from nine assembly reactionscombining each of the three loop 1 DNA fragments with each of the threeloop 4 DNA fragments as templates and the oligonucleotides Sfi-tag5′-CGGCTGAGCGGCCCAGC-3′ (SEQ ID NO:74) and Not-tag5′-GCACTCCTGCGGCCGCG-3′ (SEQ ID NO:75) as primers using standardprocedures. Each of the three loop 1 fragments is amplified in a primaryPCR reaction with pPhSP-D phagemid DNA (cf. Example 10) as template andthe oligonucleotides Sp-dloop1a fo (SEQ ID NO:76), Sp-dloop1b fo (SEQ IDNO:77)or Sp-dloop1c fo (SEQ ID NO:78) and SfiSP-D (SEQ ID NO:64) asprimers, and further amplified in a PCR reaction using Sfi-tag (SEQ IDNO:74) and Sp-dloop1-tag fo (SEQ ID NO:79) as primers. Each of the threeDNA loop 4 fragments is amplified in a primary PCR reaction with pPhSP-Dphagemid DNA (cf. Example 10) as template and the oligonucleotidesSp-dloop4a rev (SEQ ID NO:81), Sp-dloop4b rev (SEQ ID NO:82) orSp-dloop4c rev (SEQ ID NO:83) and NotSP-D (SEQ ID NO:65) as primersusing standard procedures and further amplified in a PCR reaction usingSp-dloop4-tag rev (SEQ ID NO:80) and Not-tag (SEQ ID NO:75) as primers.In the oligonucleotide sequences N denotes a mixture of 25% of each ofthe nucleotides T, C, G, and A, respectively, and S denotes a mixture of50% of C and G, encoding the appropriately randomized nucleotidesequence. The ligation mixture is used to transform so-calledelectrocompetent E. coli TG-1 cells by electroporation using standardprocedures. After transformation the E. coli TG-1 cells are plated on2×TY-agar plates containing 0.2 mg ampicillin/mL and 20 glucose andincubated over night at 30° C.

EXAMPLE 13

Construction of the Phage Library PhtCTLD-lb004

All oligonucleotides used in this example were supplied by DNATechnology (Aarhus, Denmark).

The phage library PhtCTLD-lb004, containing random amino acid residuescorresponding to PhtCTLD (SEQ ID NO:15) positions 97 to 102 or 98 to101(loop 3) and positions 116 to 122 or 118 to 120 (loop 5) wasconstructed by ligation of 20 μg KpnI and MunI restricted pPhtCTLDphagemid DNA (cf. Example 1) with 10 μg of a KpnI and MunI restrictedDNA fragment population encoding the randomized loop 3 and 5 regions.The DNA fragment population was amplified from nine primary PCRreactions combining each of the three loop 3 DNA fragments with each ofthe three loop 5 DNA fragments. The fragments was amplified with eitherof the oligonucleotides loop3a (SEQ ID NO:84), loop3b (SEQ ID NO: 85),or loop3c (SEQ ID NO:86) as template and loop5a(SEQ ID NO:87),loop5b(SEQ ID NO:88)or loop5c(SEQ ID NO:89) and loop3-4rev(SEQ ID NO:91)as primers. The DNA fragments were further amplified in PCR reactions,using the primary PCR product as template and the oligonucleotideloop3-4rev (SEQ ID NO:91) and loop3-4-stag fo (SEQ ID NO:90) as primers.All PCR reactions were performed using standard procedures.

In the oligonucleotide sequences N denotes a mixture of 25% of each ofthe nucleotides T, C, G, and A, respectively and S denotes a mixture of50% of C and G, encoding the appropriately randomized nucleotidesequence. The ligation mixture was used to transform so-calledelectrocompetent E. coli TG-1 cells by electroporation using standardprocedures. After transformation the E. coli TG-1 cells were plated on2×TY-agar plates containing 0.2 mg ampicillin/mL and 20 glucose andincubated over night at 30° C.

The size of the resulting library, PhtCTLD-lb004, was determined to7*10⁹ clones. Sixteen clones from the library were picked and phagemidDNA isolated. The nucleotide sequence of the loop-regions weredetermined (DNA Technology, Aarhus, Denmark). Thirteen clones were foundto contain correct loop inserts and three clones contained a frameshiftmutation in the region.

EXAMPLE 14

Selection of Phtlec-Phages and PhtCTLD-Phages Binding to the Blood GroupA Sugar Moiety Immobilised on Human Serum Albumin

Phages grown from glycerol stocks of the libraries Phtlec-lb001 andPhtlec-lb002 (cf. Example 4) and phages grown from a glycerol stock ofthe library PhtCTLD-lb003 (cf. Example 5), using a standard procedure,were used in an experiment designed for the selection of Phtlec- andPhtCTLD derived phages with specific affinity to the blood group A sugarmoiety immobilized on human serum albumin, A-HA, by panning in 96-wellMaxisorb micro-titerplates (NUNC, Denmark) using standard procedures.

Initially, the phage supernatants were precipitated with 0.3 volume of asolution of 20% polyethylene glycol 6000 (PEG) and 2.5 M NaCl, and thepellets re-suspended in TE-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA).After titration on E. coli TG-1 cells, phages derived from Phtlec-lb001and -lb002 were mixed (#1) in a 1:1 ratio and adjusted to 5*10¹² pfu/mLin 2*TY medium, and phages grown from the PhtCTLD-lb003 library (#4)were adjusted to 2.5*10¹² pfu/mL in 2*TY medium.

One microgram of the “antigen”, human blood group A trisaccharideimmobilised on human serum albumin, A-HA, (Glycorex AB, Lund, Sweden) in100 μL PBS (PBS, 0.2 g KCl, 0.2 g KH₂PO₄, 8 g NaCl, 1.44 g Na₂HPO₄,2H₂O, water to 1 L, and adjusted to pH 7.4 with NaOH), in each of threewells, was coated over night at 4° C. and at room temperature for onehour, before the first round of panning. After washing once with PBS,wells were blocked with 300 μL PBS and 3% non fat dried milk for onehour at room temperature. After blocking wells were washed once in PBSand 0.1% Tween 20 and three times with PBS before the addition of amixture of 50 μL of the phage suspension and 50 μL PBS, 6% non fat driedmilk. The phages were allowed to bind at room temperature for two hoursbefore washing eight times with PBS, Tween 20, and eight times with PBS.Bound phages were eluted from each well by trypsin digestion in 100 μL(1 mg/mL trypsin in PBS) for 30 min. at room temperature, and used forinfection of exponentially growing E. coli TG1 cells before plating andtitration on 2×TY agar plates containing 20 glucose and 0.1 mg/mLampicillin.

In the second round of selection, 150 μL of crude phage supernatant,grown from the first round output colonies, was mixed with 150 μL PBS,6% non fat dried milk, and used for panning distributing 100 μL of themixture in each of three A-HA coated wells, as previously described.Stringency in binding was increased by increasing the number of washingsteps from 16 to 32. 300 μL of phage mixture was also used for panningin three wells, which had received no antigen as control.

In the third round of selection, 150 μL of crude phage supernatant,grown from the second round output colonies, was mixed with 150 μL PBS,6% non fat dried milk, and used for panning distributing 100 μL of themixture in each of three A-HA coated wells, as previously described. Thenumber of washing steps was again 32. 300 μL of phage mixture was alsoused for panning in three wells, which had received no antigen ascontrol.

The results from the selection procedure are summarised in Table 8

TABLE 8 Selection of Phtlec phages (#1) and PhtCTLD phages (#4) bindingto A-HA by panning and elution with trypsin digestion. A-HA Blank RatioRound 1 #1 0.8 * 10³ n.a. n.a. #4 1.1 * 10³ n.a. n.a. Round 2 #1 1.0 *10³ 0.5 * 10² 20 #4 1.3 * 10³ 0.5 * 10² 26 Round 3 #1 8.0 * 10⁴ 0.5 *10² 1600 #4 9.0 * 10⁵ 0.5 * 10² 18000 n.a. not applicable.

48 clones from each of the #1 and #4 series were picked and grown in a96 well microtiter tray and phages produced by infection with M13K07helper phage using a standard procedure. Phages from the 96 phagesupernatants were analyzed for binding to the A-HA antigen and fornon-specific binding to hen egg white lysozyme using an ELISA-typeassay. Briefly, in each well 1 μg of A-HA in 100 μL PBS (PBS, 0.2 g KCl,0.2 g KH₂PO₄, 8 g NaCl, 1.44 g Na₂HPO₄, 2H₂O, water to 1 L, and adjustedto pH 7.4 with NaOH) or 1 μg of hen egg white lysozyme in 100 μL PBS(for analysis of non specific binding) was used for over night coatingat 4° C. and at room temperature for one hour. After washing once withPBS, wells were blocked with 300 μL PBS and 3% non fat dried milk forone hour at room temperature. After blocking wells were washed once inPBS and 0.1% Tween 20 and three times with PBS before the addition of 50μL phage supernatant in 50 μL PBS, 6% non fat dried milk. The phagemixtures were allowed to bind at room temperature for two hours beforewashing three times with PBS, Tween 20, and three times with PBS. Afterwashing, 50 μL of a 1:5000 dilution of a HRP-conjugated anti-gene VIIIantibody (Amersham Pharmacia Biotech) in PBS, 3% non fat dried milk, wasadded to each well and incubated at room temperature for one hour. Afterbinding of the “secondary” antibody wells were washed three times withPBS, Tween 20, and three times with PBS before the addition of 50 μL ofTMB substrate (DAKO-TMB One-Step Substrate System, DAKO, Denmark).Reaction was allowed to proceed for 20 min. before quenching with 0.5 MH₂SO₄, and analysis. The result of the ELISA analysis showed “hits” interms of specific binding to A-HA of phages in both series (FIGS. 34 and35), as judged by a signal ratio between signal on A-HA to signal onlysozyme at or above 1.5, and with a signal above background.

From the #1 series 13 hits were identified and 28 hits were identifiedfrom the #4 series.

REFERENCES

Aspberg, A., Miura, R., Bourdoulous, S., Shimonaka, M., Heinegård, D.,Schachner, M., Ruoslahti, E., and Yamaguchi, Y. (1997). “The C-typelectin domains of lecticans, a family of aggregating chondroitin sulfateproteoglycans, bind tenascin-R by protein-protein interactionsindependent of carbohydrate moiety”. Proc. Natl. Acad. Sci. (USA) 94:10116-10121

Bass, S., Greene, R., and Wells, J. A. (1990). “Hormone phage: anenrichment method for variant proteins with altered binding properties”.Proteins 8: 309-314

Benhar, I., Azriel, R., Nahary, L., Shaky, S., Berdichevsky, Y.,Tamarkin, A., and Wels, W. (2000). “Highly efficient selection of phageantibodies mediated by display of antigen as Lpp-OmpA' fusions on livebacteria”. J. Mol. Biol. 301: 893-904

Berglund, L. and Petersen, T. E. (1992). “The gene structure oftetranectin, a plasminogen binding protein”. FEBS Letters 309: 15-19

Bertrand, J. A., Plgnol, D., Bernard, J-P., Verdier, J-M., Dagorn, J-C.,and Fontecilla-Camps, J. C. (1996). “Crystal structure of humanlithostathine, the pancreatic inhibitor of stone formation”. EMBO J. 15:2678-2684

Bettler, B., Texido, G., Raggini, S., Ruegg, D., and Hofstetter, H.(1992). “Immunoglobulin E-binding site in Fc epsilon receptor (Fcepsilon RII/CD23) identified by homolog-scanning mutagenesis”. J. Biol.Chem. 267: 185-191

Blanck, O., Iobst, S. T., Gabel, C., and Drickamer, K.(1996).“Introduction of selectin-like binding specificity into ahomologous mannose-binding protein”. J. Biol. Chem. 271: 7289-7292

Boder, E. T. and Wittrup, K. D. (1997). “Yeast surface display forscreening combinatorial polypeptide libraries”. Nature Biotech. 15:553-557

Burrows L, Iobst S T, Drickamer K. (1997) “Selective binding ofN-acetylglucosamine to the chicken hepatic lectin”. Biochem J.324:673-680

Chiba, H., Sano, H., Saitoh, M., Sohma, H., Voelker, D. R., Akino, T.,and Kuroki, Y. (1999). “Introduction of mannose binding protein-typephosphatidylinositol recognition into pulmonary surfactant protein A”.Biochemistry 38: 7321-7331

Christensen, J. H., Hansen, P. K., Lillelund, O., and Thogersen, H. C.(1991). “Sequence-specific binding of the N-terminal three-fingerfragment of Xenopus transcription factor IIIA to the internal controlregion of a 5S RNA gene”. FEBS Letters 281: 181-184

Cyr, J. L. and Hudspeth, A. J. (2000). “A library ofbacteriophage-displayed antibody fragments directed against proteins ofthe inner ear”. Proc. Natl. Acad. Sci (USA) 97: 2276-2281

Drickamer, K. (1992). “Engineering galactose-binding activity into aC-type mannose-binding protein”. Nature 360: 183-186

Drickamer, K. and Taylor, M. E. (1993). “Biology of animal lectins”.Annu. Rev. Cell Biol. 9: 237-264

Drickamer, K. (1999). “C-type lectin-like domains”. Curr. Opinion Struc.Biol. 9: 585-590

Dunn, I. S. (1996). “Phage display of proteins”. Curr. Opinion Biotech.7: 547-553

Erbe, D. V., Lasky, L. A., and Presta, L. G. “Selectin variants”. U.S.Pat. No. 5,593,882

Ernst, W. J., Spenger, A., Toellner, L., Katinger, H.,Grabherr, R. M.(2000). “Expanding baculovirus surface display. Modification of thenative coat protein gp64 of Autographa californica NPV”. Eur. J.Biochem. 267: 4033-4039

Ewart, K. V., Li, Z., Yang, D. S. C., Fletcher, G. L., and Hew, C. L.(1998). “The ice-binding site of Atlantic herring antifreeze proteincorresponds to the carbohydrate-binding site of C-type lectins”.Biochemistry 37: 4080-4085

Feinberg, H., Park-Snyder, S., Kolatkar, A. R., Heise, C. T., Taylor, M.E., and Weis, W. I. (2000). “Structure of a C-type carbohydraterecognition domain from the macrophage mannose receptor”. J. Biol. Chem.275: 21539-21548

Fujii, I., Fukuyama, S., Iwabuchi, Y., and Tanimura, R. (1998).“Evolving catalytic antibodies in a phage-displayed combinatoriallibrary”. Nature Biotech. 16: 463-467

Gates, C. M., Stemmer, W. P. C., Kaptein, R., and Schatz, P. J. (1996).“Affinity selective isolation of ligands from peptide libraries throughdisplay on a lac repressor “headpiece dimer”. J. Mol. Biol. 255: 373-386

Graversen, J. H., Lorentsen, R. H., Jacobsen, C., Moestrup, S. K.,Sigurskjold, B. W., Thogersen, H. C., and Etzerodt, M. (1998). “Theplasminogen binding site of the C-type lectin tetranectin is located inthe carbohydrate recognition domain, and binding is sensitive to bothcalcium and lysine”. J. Biol. Chem. 273:29241-29246

Graversen, J. H., Jacobsen, C., Sigurskjold, B. W., Lorentsen, R. H.,Moestrup, S. K., Thogersen, H. C., and Etzerodt, M. (2000). “MutationalAnalysis of Affinity and Selectivity of Kringle-Tetranectin Interaction.Grafting novel kringle affinity onto the tetranectin lectin scaffold”.J. Biol. Chem. 275: 37390-37396

Griffiths, A. D. and Duncan, A. R. (1998). “Strategies for selection ofantibodies by phage display”. Curr. Opinion Biotech. 9: 102-108

Holtet, T. L., Graversen, J. H., Clemmensen, I., Thogersen, H. C., andEtzerodt, M. (1997). “Tetranectin, a trimeric plasminogen-binding C-typelectin”. Prot. Sci. 6: 1511-1515

Honma, T., Kuroki, Y., Tzunezawa, W., Ogasawara, Y., Sohma, H., Voelker,D. R., and Akino, T. (1997). “The mannose-binding protein A region ofglutamic acid185-alanine221 can functionally replace the surfactantprotein A region of glutamic acid195-phenylalanine228 without loss ofinteraction with lipids and alveolar type II cells”. Biochemistry 36:7176-7184

Huang, W., Zhang, Z., and Palzkill, T. (2000). “Design of potentbeta-lactamase inhibitors by phage display of beta-lactamase inhibitoryprotein”. J. Biol. Chem. 275: 14964-14968

Hufton, S. E., van Neer, N., van den Beuken, T., Desmet, J., Sablon, E.,and Hoogenboom, H. R. (2000). “Development and application of cytotoxicT lymphocyte-associated antigen 4 as a protein scaffold for thegeneration of novel binding ligands”. FEBS Letters 475: 225-231

Håkansson, K., Lim, N. K., Hoppe, H-J., and Reid, K. B. M. (1999).“Crystal structure of the trimeric alpha-helical coiled-coil and thethree lectin domains of human lung surfactant protein D”. StructureFolding and Design 7: 255-264

Iobst, S. T., Wormald, M. R., Weis, W. I., Dwek, R. A., and Drickamer,K. (1994). “Binding of sugar ligands to Ca(2+)-dependent animal lectins.I. Analysis of mannose binding by site-directed mutagenesis and NMR”. J.Biol. Chem. 269: 15505-15511

Iobst, S. T. and Drickamer, K. (1994). “Binding of sugar ligands toCa(2+)-dependent animal lectins. II. Generation of high-affinitygalactose binding by site-directed mutagenesis”. J. Biol. Chem. 269:15512-15519

Iobst, S. T. and Drickamer, K. (1996). “Selective sugar binding to thecarbohydrate recognition domains of the rat hepatic and macrophageasialoglycoprotein receptors”. J. Biol. Chem. 271: 6686-6693

Jaquinod, M., Holtet, T. L., Etzerodt, M., Clemmensen, I., Thogersen, H.C., and Roepstorff, P. (1999). “Mass Spectrometric Characterisation ofPost-Translational Modification and Genetic Variation in HumanTetranectin”. Biol. Chem. 380: 1307-1314

Kastrup, J. S., Nielsen, B. B., Rasmussen, H., Holtet, T. L., Graversen,J. H., Etzerodt, M., Thogersen, H. C., and Larsen, I. K. (1998).“Structure of the C-type lectin carbohydrate recognition domain of humantetranectin”. Acta. Cryst. D 54: 757-766

Kogan, T. P., Revelle, B. M., Tapp, S., Scott, D., and Beck, P. J.(1995). “A single amino acid residue can determine the ligandspecificity of E-selectin”. J. Biol. Chem. 270: 14047-14055

Kolatkar, A. R., Leung, A. K., Isecke, R., Brossmer, R., Drickamer, K.,and Weis, W. I. (1998). “Mechanism of N-acetylgalactosamine binding to aC-type animal lectin carbohydrate-recognition domain”. J. Biol. Chem.273: 19502-19508

Lorentsen, R. H., Graversen, J. H., Caterer, N. R., Thogersen, H. C.,and Etzerodt, M. (2000). “The heparin-binding site in tetranectin islocated in the N-terminal region and binding does not involve thecarbohydrate recognition domain”. Biochem. J. 347: 83-87

Marks, J. D., Hoogenboom, H. R., Griffiths, A. D., and Winter, G.(1992). “Molecular evolution of proteins on filamentous phage. Mimickingthe strategy of the immune system”. J. Biol. Chem. 267: 16007-16010

Mann K, Weiss I M, Andre S, Gabius H J, Fritz M. (2000). “The amino-acidsequence of the abalone (Haliotis laevigata) nacre protein perlucin.Detection of a functional C-type lectin domain with galactose/mannosespecificity”. Eur. J. Biochem. 267: 5257-5264

McCafferty, J., Jackson, R. H., and Chiswell, D. J. (1991).“Phage-enzymes: expression and affinity chromatography of functionalalkaline phosphatase on the surface of bacteriophage”. Prot. Eng. 4:955-961

McCormack, F. X., Kuroki, Y., Stewart, J. J., Mason, R. J., and Voelker,D. R. (1994). “Surfactant protein A amino acids Glu195 and Arg197 areessential for receptor binding, phospholipid aggregation, regulation ofsecretion, and the facilitated uptake of phospholipid by type II cells”.J. Biol. Chem. 269: 29801-29807

McCormack, F. X., Festa, A. L., Andrews, R. P., Linke, M., and Walzer,P. D. (1997). “The carbohydrate recognition domain of surfactant proteinA mediates binding to the major surface glycoprotein of Pneumocystiscarinii”. Biochemistry 36: 8092-8099

Meier, M., Bider, M. D., Malashkevich, V. N., Spiess, M., and Burkhard,P. (2000). “Crystal structure of the carbohydrate recognition domain ofthe H1 subunit of the asialoglycoprotein receptor”. J. Mol. Biol. 300:857-865

Mikawa, Y. G., Maruyama, I. N., and Brenner, S. (1996). “Surface displayof proteins on bacteriophage lambda heads”. J. Mol. Biol. 262: 21-30

Mio H, Kagami N, Yokokawa S, Kawai H, Nakagawa S, Takeuchi K, Sekine S,Hiraoka A. (1998). “Isolation and characterization of a cDNA for humanmouse, and rat full-length stem cell growth factor, a new member ofC-type lectin superfamily”. Biochem. Biophys. Res. Commun. 249: 124-130

Mizuno, H., Fujimoto, Z., Koizumi, M., Kano, H., Atoda, H., and Morita,T. (1997). “Structure of coagulation factors IX/X-binding protein, aheterodimer of C-type lectin domains”. Nat. Struc. Biol. 4: 438-441

Ng, K. K., Park-Snyder, S., and Weis, W. I. (1998a). “Ca²⁺-dependentstructural changes in C-type mannose-binding proteins”. Biochemistry 37:17965-17976

Ng, K. K. and Weis, W. I. (1998b). “Coupling of prolyl peptide bondisomerization and Ca2+ binding in a C-type mannose-binding protein”.Biochemistry 37: 17977-17989

Nielsen, B. B., Kastrup, J. S., Rasmussen, H., Holtet, T. L., Graversen,J. H., Etzerodt, M., Thogersen, H. C., and Larsen, I. K. (1997).“Crystal structure of tetranectin, a trimeric plasminogen-bindingprotein with an alpha-helical coiled coil”. FEBS Letters 412: 388-396

Nissim A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C.,Lane, D., and Winter, G. (1994). “Antibody fragments from a ‘single pot’phage display library as immunochemical reagents”. EMBO J. 13: 692-698

Ogasawara, Y. and Voelker, D. R. (1995). “Altered carbohydraterecognition specificity engineered into surfactant protein D revealsdifferent binding mechanisms for phosphatidylinositol andglucosylceramide”. J. Biol. Chem. 270: 14725-14732

Ohtani, K., Suzuki, Y., Eda, S., Takao, K., Kase, T., Yamazaki, H.,Shimada, T., Keshi, H., Sakai, Y., Fukuoh, A., Sakamoto, T., andWakamiya, N. (1999). “Molecular cloning of a novel human collectin fromliver (CL-L1)”. J. Biol. Chem. 274: 13681-13689

Pattanajitvilai, S., Kuroki, Y., Tsunezawa, W., McCormack, F. X., andVoelker, D. R. (1998). “Mutational analysis of Arg197 of rat surfactantprotein A. His197 creates specific lipid uptake defects”. J. Biol. Chem.273: 5702-5707

Poget, S. F., Legge, G. B., Proctor, M. R., Butler, P. J., Bycroft, M.,and Williams, R. L. (1999). “The structure of a tunicate C-type lectinfrom Polyandrocarpa misakiensis complexed with D-galactose”. J. Mol.Biol. 290: 867-879

Revelle, B. M., Scott, D., Kogan, T. P., Zheng, J., and Beck, P. J.(1996). “Structure-function analysis of P-selectin-sialyl LewisX bindinginteractions. Mutagenic alteration of ligand binding specificity”. J.Biol. Chem. 271: 4289-4297

Sano, H., Kuroki, Y., Honma, T., Ogasawara, Y., Sohma, H., Voelker, D.R., and Akino, T. (1998). “Analysis of chimeric proteins identifies theregions in the carbohydrate recognition domains of rat lung collectinsthat are essential for interactions with phospholipids, glycolipids, andalveolar type II cells”. J. Biol. Chem. 273: 4783-4789

Schaffitzel, C., Hanes, J., Jermutus, L., and Placktun, A. (1999).“Ribosome display: an in vitro method for selection and evolution ofantibodies from libraries”. J. Immunol. Methods 231: 119-135

Sheriff, S., Chang, C. Y., and Ezekowitz, R. A. (1994). “Humanmannose-binding protein carbohydrate recognition domain trimerizesthrough a triple alpha-helical coiled-coil”. Nat. Struc. Biol. 1:789-794

Sørensen, C. B., Berglund, L., and Petersen, T. E. (1995). “Cloning of acDNA encoding murine tetranectin”. Gene 152: 243-245

Torgersen, D., Mullin, N. P., and Drickamer, K. (1998). “Mechanism ofligand binding to E- and P-selectin analyzed usingselectin/mannose-binding protein chimeras”. J. Biol. Chem. 273:6254-6261

Tormo, J., Natarajan, K., Margulies, D. H., and Mariuzza, R. A. (1999).“Crystal structure of a lectin-like natural killer cell receptor boundto its MHC class I ligand”. Nature 402: 623-631

Tsunezawa, W., Sano, H., Sohma, H., McCormack, F. X., Voelker, D. R.,and Kuroki, Y. (1998). “Site-directed mutagenesis of surfactant proteinA reveals dissociation of lipid aggregation and lipid uptake by alveolartype II cells”. Biochim. Biophys. Acta 1387: 433-446

Weis, W. I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W. A.(1991). “Structure of the calcium-dependent lectin domain from a ratmannose-binding protein determined by MAD phasing”. Science 254:1608-1615

Weis, W. I., and Drickamer, K. (1996). “Structural basis oflectin-carbohydrate recognition”. Annu. Rev. Biochem. 65: 441-473

Whitehorn, E. A., Tate, E., Yanofsky, S. D., Kochersperger, L., DavisA., Mortensen, R. B., Yonkovic, S., Bell, K., Dower, W. J., and Barrett,R. W. (1995). “A generic method for expression and use of “tagged”soluble versions of cell surface receptors“. Bio/Technology 13:1215-1219

Wragg, S. and Drickamer, K. (1999). “Identification of amino acidresidues that determine pH dependence of ligand binding to theasialoglycoprotein receptor during endocytosis”. J. Biol. Chem. 274:35400-35406

Zhang, H., Robison, B., Thorgaard, G. H., and Ristow, S. S. (2000).“Cloning, mapping and genomic organization of a fish C-type lectin genefrom homozygous clones of rainbow trout (Oncorhynchos Mykiss)”. Biochim.et Biophys. Acta 1494: 14-22

1-76. (canceled)
 77. A combinatorial library comprising an ensemble ofvariant C-type lectin-like domain (CTLD) polypeptides having thescaffold structure of a CTLD polypeptide and a randomized CTLD loopregion, the CTLD scaffold comprising the following structural elements:five β-strands and two α-helices sequentially appearing in the order β1,α1, α2, β2, β3, β4, and β5, the β-strands being arranged in twoanti-parallel β-sheets, one β-sheet composed of β1 and β5, the otherβ-sheet composed of β2, β3, and β4, and at least two disulfide bridges,one disulfide bridge connecting α1 and β5 and one disulfide bridgeconnecting β3 and a polypeptide segment connecting β4 and β5; and therandomized CTLD loop region consisting of two loop polypeptide segments,loop segment A (LSA) connecting β2 and β3, and loop segment B (LSB)connecting β3 and β4, wherein the amino acid sequence of LSA and/or LSBis randomized from the amino acid sequence of a wildtype CTLD loopregion by random amino acid substitution, deletion, insertion, or anycombination thereof of the wildtype CTLD loop sequence.
 78. Thecombinatorial library of claim 77, wherein the CTLD is selected from ahuman tetranectin (hTN), mannose binding protein (MBP), surfactantprotein D (SP-D), LY49A NK receptor domain (LY49A), asidoglycoproteinreceptor (Hl-ASR), mouse macrophage receptor (MMR-4), Factor 1X/Xbinding protein A (IX-A), Factor 1X/X binding protein B (IX-B),lithostatin (Lit), tunicate C-type lectin (TU14) CTLD, and mousetetranectin (mTN) CTLD.
 79. The combinatorial library of claim 77,wherein the polypeptides further comprise N-terminal and/or C-terminalextensions of the CTLD.
 80. The combinatorial library of claim 79,wherein the N-terminal and/or C-terminal extensions contain effector,enzyme, further binding and/or multimerizing functions.
 81. Thecombinatorial library of claim 79, wherein the N-terminal and/orC-terminal extensions are the non-CTLD-portions of a native C-typelectin-like protein or a C-type lectin or a C-type lectin lacking afunctional transmembrane domain.
 82. The combinatorial library of claim77, wherein the amino acid residues differ between different members ofthe ensemble of polypeptides in at least at two amino acid sequencepositions in the randomized loop region.
 83. The combinatorial libraryof claim 82, wherein at least three of the amino acid sequence positionsof Loop Segment A are randomized.
 84. The combinatorial library of claim82, wherein at least two of the amino acid sequence positions of LoopSegment B are randomized.
 85. The combinatorial library of claim 77,wherein the amino acid residues differ between different members of theensemble of polypeptides at least at one amino acid sequence position inthe Loop Segment A and at least at one amino acid sequence position inLoop Segment B.
 86. The combinatorial library of claim 77, wherein theamino acid residues differ between different members of the ensemble ofpolypeptides at any one or more sequence positions in the loop regioncorresponding to amino acid residues 72-107 and 114-117 of SEQ ID NO:276; amino acid residues 66-99 and 105-107 of SEQ ID NO: 277; amino acidresidues 69-102 and 108-110 of SEQ ID NO: 278; amino acid residues 72-93and 99-100 of SEQ ID NO: 279; amino acid residues 62-101 and 107-109 ofSEQ ID NO: 280; amino acid residues 77-111 and 117-121 of SEQ ID NO:281; amino acid residues 71-100 and 105-112of SEQ ID NO: 282; amino acidresidues 68-94 and 99-104 of SEQ ID NO: 283; amino acid residues 68-103and 111-117 of SEQ ID NO: 284; amino acid residues 54-94 and 100-103 ofSEQ ID NO: 285; and amino acid residues 115-151 and 158-161 of SEQ IDNO:
 289. 87. The combinatorial library of claim 86, wherein the aminoacid residues differ between different members of the ensemble ofpolypeptides at any one or more sequence positions in the loop regioncorresponding to amino acid residues 72-79, 81-85, 91-99, 101-107, and114-117 of SEQ ID NO:
 276. 88. The combinatorial library of claim 86,wherein the amino acid residues differ at any of the sequence positionscorresponding to 73-78, 93-98, 102-105, and 114-117 of SEQ ID NO: 276.89. The combinatorial library of claim 86, wherein the amino acidresidues differ at any of the sequence positions corresponding to 73-75,77-78, and 102-105 of SEQ ID NO:
 276. 90. The combinatorial library ofclaim 86, wherein the amino acid residues differ at any of the sequencepositions corresponding to 93-98 and 102-105 of SEQ ID NO:
 276. 91. Thecombinatorial library of claim 86, wherein the amino acid residuesfurther differ at the sequence position corresponding to120 of SEQ IDNO:
 276. 92. The combinatorial library of claim 86, wherein the aminoacid residues differ at any of the sequence positions corresponding to93-98 and 114-117 of SEQ ID NO:
 276. 93. The combinatorial library ofclaim 88, wherein the amino acid residues further differ at any of thesequence positions corresponding to 112, 113, and 118 of SEQ ID NO: 276.94. The combinatorial library of claim 86, wherein the amino acidresidues differ at any of the sequence positions corresponding to 94-97and 114-116 of SEQ ID NO:
 276. 95. The combinatorial library of claim94, wherein the amino acid residues differ at any of the sequencepositions corresponding to 73-75, 77-78, and 104-105 of SEQ ID NO: 276.96. The combinatorial library of claim 86, wherein the amino acidresidues differ at any one or more sequence positions in the loop regioncorresponding to amino acid residues 66-73, 75-79, 85-90, 92-99, and105-107 of SEQ ID NO:
 277. 97. The combinatorial library of claim 86,wherein the amino acid residues differ at any one or more sequencepositions in the loop region corresponding to amino acid residues 69-76,78-82, 88-93, 95-102, and 108-110 of SEQ ID NO:
 278. 98. Thecombinatorial library of claim 77, wherein 1-10 amino acid residues aresubstituted, deleted, or inserted in any one or more of the α-helices,β-strands, and connecting segments.
 99. The combinatorial library ofclaim 77, wherein the 1-10 amino acid residues are substituted, deleted,or inserted in any one or more of the β2, β3, and β4-strands.
 100. Thecombinatorial library of claim 77, wherein the polypeptide sequenceoutside of the loop region is at least 95% identical to the amino acidsequence outside the loop region of one of SEQ ID NO:276, 277 and 278.101. A combinatorial library comprising an ensemble of polypeptidescomprising an amino acid sequence at least 95% identical to amino acids1-71, and 114-117 of SEQ ID NO:276 wherein one or more of amino acids72-79, 81-85, 91-99, 101-107, and 114-117 are randomized.
 102. A nucleicacid library comprising a multitude of nucleic acids encoding variantC-type lectin-like domain (CTLD) polypeptides having the scaffoldstructure of a CTLD polypeptide and a randomized CTLD loop region, theCTLD scaffold comprising the following structural elements: fiveβ-strands and two α-helices sequentially appearing in the order β1, α1,α2, β2, β3, β4, and β5, the (β-strands being arranged in twoanti-parallel (β-sheets, one (3-sheet composed of β1 and β5, the other(3-sheet composed of β2, β3, and β4, and at least two disulfide bridges,one disulfide bridge connecting α1 and β5 and one disulfide bridgeconnecting β3 and a polypeptide segment connecting β4 and β5; and therandomized CTLD loop region consisting of two loop polypeptide segments,loop segment A (LSA) connecting β2 and β3, and loop segment B (LSB)connecting β3 and β4, wherein the amino acid sequence of LSA and/or LSBis randomized from the amino acid sequence of a wildtype CTLD loopregion by random amino acid substitution, deletion, insertion, or anycombination thereof of the wildtype CTLD loop sequence.
 103. The libraryof claim 102, wherein the CTLD loop region is randomized by substitutingthe portion of the nucleic acid molecules encoding some or all of theloop regions with a nucleic acid fragment randomly selected from amultitude of nucleic acid fragments.
 104. The combinatorial library ofclaim 103, wherein the amino acid residues differ between differentmembers of the ensemble of polypeptides in at least at two amino acidsequence positions in the randomized loop region.
 105. The combinatoriallibrary of claim 103, wherein at least three of the amino acid sequencepositions of Loop Segment A are randomized.
 106. The combinatoriallibrary of claim 103, wherein at least two of the amino acid sequencepositions of Loop Segment B are randomized.
 107. The library of claim102, wherein the nucleotide sequence encoding the polypeptide sequenceoutside of the loop region is altered to facilitate the excision of partor all of the loop region and the insertion of an altered looppolypeptide sequence while the scaffold structureof the CTLD issubstantially maintained.
 108. A method of preparing the combinatoriallibrary based upon a CTLD scaffold structure, the method comprising: (a)inserting in a suitable vector a nucleic acid encoding variant C-typelectin-like domain (CTLD) polypeptide having the scaffold structure of aCTLD polypeptide and a randomized CTLD loop region, the CTLD scaffoldcomprising the following structural elements: five β-strands and twoα-helices sequentially appearing in the order β1, α1, α2, β2, β3, β4,and β5, the (β-strands being arranged in two anti-parallel (β-sheets,one (β-sheet composed of β1 and β5, the other (β-sheet composed of β2,β3, and β4, and at least two disulfide bridges, one disulfide bridgeconnecting α1 and β5 and one disulfide bridge connecting β3 and apolypeptide segment connecting β4 and β5; and the randomized CTLD loopregion consisting of two loop polypeptide segments, loop segment A (LSA)connecting β2 and β3, and loop segment B (LSB) connecting β3 and β4,wherein the amino acid sequence of LSA and/or LSB is randomized from theamino acid sequence of a wildtype CTLD loop region by random amino acidsubstitution, deletion, insertion, or any combination thereof of thewildtype CTLD loop sequence, (b) optionally introducing restrictionendonuclease recognition sites, the recognition sites being properlylocated in the sequence at or close to the ends of the sequence encodingthe loop region of the CTLD or part thereof, (c) excising the DNAfragment encoding the loop region or part thereof using restrictionendonucleases; (d) ligating randomized mixtures of DNA fragments intothe loop region of the restricted vector, and inducing the vector toexpress randomized polypeptides having the scaffold structure of theCTLD and a randomized loop region in a suitable medium.